Transfusion Update

Indian Society of Blood Transfusion and Immunohaematology (ISBTI)

Transfusion Update
Editor-in-Chief
Kanchan Bhardwaj
MD MAMS FIAC FIMSA
Professor and Head
Department of Transfusion Medicine
Government Medical College, Patiala, Punjab, India

Associate Editors
BL Bhardwaj
MD MAMS FCCP FIMSA FIACP FNCCP FGSI FIACM
Professor
Department of Medicine
Government Medical College
Patiala, Punjab, India

Kusum K Thakur
MD PGDMCH PGDHHM MISBTI MIMA MAATM
Lecturer
Department of Transfusion Medicine
Government Medical College
Patiala, Punjab, India
Secretary, ISBTI (Punjab Chapter)

Rajni Bassi
MD MISBTI MIMA
Lecturer
Department of Transfusion Medicine
Government Medical College
Patiala, Punjab, India

Assistant Editor
Harnoor Singh Bhardwaj  MBBS
Junior Resident
Government Medical College
Patiala, Punjab, India

Forewords
KK Talwar
TR Raina
Neelam Marwaha

An ISBTI Publication

The Health Sciences Publisher

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Transfusion Update

First Edition: 2015

ISBN: 978-93-5152-598-1
Printed at
 Dedicated to
My loving parents
Mrs Surjit Saini and Er BK Saini
and my teachers
 Contributors
A Surekha Devi  MD Anupam Verma  MD PDCC Bimal Kumar Agrawal  MD
Consultant and Head Additional Professor Professor and Head
Department of Transfusion Medicine Department of Transfusion Medicine Department of Medicine
Global Hospitals Sanjay Gandhi Postgraduate Institute MM Institute of Medical Sciences
Hyderabad, Andhra Pradesh, India of Medical Sciences (SGPGIMS) and Research
surekhadevi@gmail.com Lucknow, Uttar Pradesh, India Ambala, Haryana, India
aver2211@gmail.com aver@sgpgi.ac.in bkagrawal2001@yahoo.com
Aastha Miranpuri  MD
Rochester, New York, USA BK Rana 
Aradhna Sharma  MD PhD
aasthamiranpuri@gmail.com Joint Director
Medical Officer
Department of Transfusion Medicine National Accreditation Board for Hospitals and
Achhar Singh  MD Healthcare Providers (NABH) Quality Council
Assistant Professor Government Medical College
Patiala, Punjab, India of India
Department of Pulmonary Medicine New Delhi, India
Gian Sagar Medical College asharma130798@gmail.com
bkrana@nabh.co
Banur, Patiala, Punjab, India
kusum_gmcbb@yahoo.com Arun Bansal  MS MCh (Neurosurgery) BL Bhardwaj 
Neurosurgeon
MD MAMS FCCP FIMSA FIACP FNCCP FGSI FIACM
Ajata Shatru Kapoor  MS MCh (Plastic Surgery) Amar Hospital
Professor
Assistant Professor Patiala, Punjab, India
Department of Medicine
Department of General Surgery drarunneuro@gmail.com
Government Medical College
MM Institute of Medical Sciences and
Patiala, Punjab, India
Research, Ambala, Haryana, India Arvind Chahal  MBBS bachan_bhardwaj@yahoo.co.in
kapoorajatashatru@yahoo.com Junior Resident
Department of Medicine Devinder Singh Sandhu
Ajay Bahl  MD DM
Pt BD Sharma Postgraduate Institute MD (Medicine) DM (Medical Oncology)
Consultant
of Medical Sciences Consultant
Department of Cardiology
Rohtak, Haryana, India Oncologist and Hematologist
Postgraduate Institute of Medical Education
med.pgims@hry.nic.in Sandhu Cancer Center
and Research (PGIMER)
Chandigarh, India Ludhiana, Punjab, India
drajaybahl@hotmail.com Aseem K Tiwari  MD drsandhu@sandhucancercentre.com
Associate Director
Ajit C Gorakshakar  PhD Department of Transfusion Medicine Dharam Paul MS
Scientist E (Deputy Director) Medanta: The Medicity Hospital Civil Surgeon (Retd)
National Institute of Immunohematology Gurgaon, Haryana, India Khanna, Punjab, India
Indian Council of Medical Research aseem.tiwari@medanta.org partola@rediffmail.com
KEM Hospital Campus, Parel
Mumbai, Maharashtra, India Ashish Jain  MD Dinesh Ahluwalia  MD
ajit5678@yahoo.com Director
Assistant Professor
Dinesh Path Lab
Department of Transfusion Medicine
Alpna Thakur  MD Patiala, Punjab, India
Postgraduate Institute of Medical Education
Assistant Professor and Research (PGIMER) drdineshahluwalia@gmail.com
Department of Dermatology Chandigarh, India
Hind Institute of Medical Sciences DP Singh  MS ISS (Cleveland) FICLS
ashishjain16@gmail.com
Lucknow, Uttar Pradesh, India Professor (Surgery)
alpna.30@gmail.com Government Medical College
Ashok Sharma  MD Patiala, Punjab, India
Amit Aggarwal  MD Professor and Head dpsingh.7@gmail.com
Consultant Department of Medicine
Fortis Escorts Heart Institute Indra Gandhi Medical College Gagandeep Kaur  MD
New Delhi, India Shimla, Himachal Pradesh, India Assistant Professor
agrawalamit@gmail.com drashoksharmamd@me.com Department of Transfusion Medicine
Government Medical College and Hospital
Anupam Sachdeva  MD Bharat Singh MD Chandigarh, India
Director Consultant Pathologist and Head Regional kaurgagandeep2701@gmail.com
Pediatric Hematology-Oncology and Bone Blood Transfusion Centre
Marrow Transplantation Guru Teg Bahadur Hospital and University Hamed Bashir  MD DM
Institute for Child Health College of Medical Sciences Consultant Cardiology
Sir Ganga Ram Hospital, New Delhi, India New Delhi, India Max Healthcare Institute
anupamace@yahoo.co.in bsdirsbtc@yahoo.co.in Saket, New Delhi, India
viii  Transfusion Update
Hari Krishan Dhawan  MD Former Chairman Manjit Kaur Mohi  MD FICOG FIMSA
Assistant Professor Board of Governors Professor and Head
Department of Transfusion Medicine Medical Council of India Department of Obstetrics and Gynaecology
Postgraduate Institute of Medical Education Former President Government Medical College
and Research (PGIMER) National Academy of Medical Sciences Patiala, Punjab, India
Chandigarh, India Former Director manjitmohi@yahoo.co.in
hkdpgimer@gmail.com Postgraduate Institute of Medical Education
and Research (PGIMER), Chandigarh, India
Manoj A Kahar  MD PhD (Pursuing)
Harnoor Singh Bhardwaj  MBBS Former Professor and Head
Blood Bank
Junior Resident Department of Pathology
Department of Cardiology
Government Medical College Government Medical College
All India Institute of Medical Sciences (AIIMS)
Patiala, Punjab, India Surat, Gujarat, India
New Delhi, India
harnoorbhardwaj@yahoo.co.in manoj_kahar@yahoo.com
kktalwar@hotmail.com
Harprit Singh  MD Manuj Wadhwa  MS MCh Ortho (UK)
Kshitija Mittal MD
National Programme Officer Ranawat Joint Replacement Fellow (USA)
Department of Transfusion Medicine
DBTS, Directorate of AIDS Control Director and Head
Government Medical College and Hospital
New Delhi, India Max Elite Institute of Orthopaedics
Chandigarh, India
harprit_1@hotmail.com and Joint Replacement
drkmittal@gmail.com
Mohali, Chandigarh, India
Hemchandra Pandey MD Kulbir Kaur MD manuj.wadhwa@gmail.com
Additional Professor Director Principal
Department of Transfusion Medicine Meenu Bajpai MD
Professor (Pathology and Transfusion
Sanjay Gandhi Postgraduate Institute of Associate Professor
Medicine)
Medical Sciences (SGPGIMS) Transfusion Medicine
Punjab Institute of Medical Sciences
Lucknow, Uttar Pradesh, India Institute of Liver and Biliary Sciences
Jalandhar, Punjab, India
New Delhi, India
Hitish Narang MD drkulbirkaur@yahoo.co.in meenubajpai@hotmail.com
Senior Consultant
Kunal R Patel  DNB (Orth) D Orth meenubajpaii@gmail.com
Transfusion Medicine
Consultant
Satguru Pratap Singh (SPS) Apollo Hospital Mina Sidhu MD
MAX Hospital
Ludhiana, Punjab, India Assistant Professor
Mohali, Chandigarh, India
hitishnarang@gmail.com Department of Transfusion Medicine
drkunalpatel@gmail.com
HS Sandhu MD Government Medical College
Kusum K Thakur Jammu and Kashmir, India
Professor and Head
MD PGDMCH PGDHHM MISBTI MIMA MAATM minathapa@gmail.com
Department of Medicine
Lecturer
Government Medical College Mini Bhatnagar Sud  MD
Department of Transfusion Medicine
Patiala, Punjab, India Associate Professor
Government Medical College
harendrasandhu@yahoo.co.in Patiala, Punjab, India Department of Medicine
Secretary MM Institute of Medical Sciences and
Ishwar Chouhan MBBS
Indian Society of Blood Transfusion and Research, Ambala, Punjab, India
Junior Resident
Department of Medicine Immunohaematology (ISBTI) Mohanvir Kaur MD
Government Medical College (Punjab Chapter) Assistant Professor
Patiala, Punjab, India kusum_gmcbb@yahoo.com Department of Pathology
i.chouhan05@gmail.com M Joseph John  MD DM Government Medical College
Associate Professor Patiala, Punjab, India
Jitendra Kumar Pehalajani MBBS
Clinical Haematology, Haemato-oncology mohanvirkaur@gmail.com
Junior Resident
Department of Medicine and Bone Marrow (Stem Cell) Transplant Unit Neelam Marwaha  MD FAMS FISHTM
Pt BD Sharma Postgraduate Institute of Christian Medical College Professor and Head
Medical Sciences Ludhiana, Punjab, India Department of Transfusion Medicine
Rohtak, Haryana, India mjosephjohn@gmail.com Postgraduate Institute of Medical Education
jitnanhe@gmail.com M Joshua Daniel Jeyakumar MD and Research, Chandigarh, India
Associate Professor and Technical Expert neelam2918@yahoo.com
Kanchan Bhardwaj
MD MAMS FIAC FIMSA MISBTI MIAPM MISHTM MIMA CDC-CMA1 NACO AMS Project Nidhi Bhatnagar  MD
Professor and Head Vinayaka Missions Medical College and AMP Lead Assessor (NABH Blood Banks)
Department of Transfusion Medicine Hospital Associate Professor
Government Medical College Salem, Tamil Nadu, India Department of Transfusion Medicine
Patiala, Punjab, India drjoshuadaniel@gmail.com BJ Medical College
drkanchan_bhardwaj@yahoo.com drjoshuadaniel@yahoo.com Ahmedabad, Gujarat, India
Manisha Shrivastava MD bhatnagarnidhi@ymail.com
KK Talwar
MD DM FAMS FNA FACC FIMSA FIACS (Canada) DSc (hc) Associate Professor and Head Nidhi Mehta MD
Advisor Department of Transfusion Medicine (Secretary-Indian chapter–AATM)
Government of Punjab Bhopal Memorial Hospital and Research Consultant Transfusion Medicine
Health and Medical Education Centre Kokilaben Dhirubhai Ambani Hospital
Member Bhopal, Madhya Pradesh, India Mumbai, Maharashtra, India
Punjab Governance Reforms Commission manishasdr@gmail.com drnidhimehta@yahoo.co.in
 Contributors  ix

P Arumugam  MD Praveen C Sobti MD Rajesh Rajput 

Professor and Head Professor MD DM (Endocrinology) FICP FIACM FIMSA

Department of Transfusion Medicine Department of Pediatrics Senior Professor and Head

The Tamil Nadu Dr MGR Medical University Dayanand Medical College Department of Endocrinology
Chennai, Tamil Nadu, India Ludhiana, Punjab, India Postgraduate Institute of Medical Sciences
pothiarumugam@gmail.com parveen_c_sobti@yahoo.co.in Rohtak, Haryana, India
drrajeshrajput@outlook.com
Pankaj Malhotra  MD MAMS FICP MACP Preeti Dewakar MD
Assistant Professor Rajni Bassi  MD MISBTI MIMA
Additional Professor (Clinical Hematology) Lecturer
Department of Internal Medicine Department of Pathology
University College of Medical Sciences Department of Transfusion Medicine
Postgraduate Institute of Medical Education Government Medical College
and Research (PGIMER) New Delhi, India
Patiala, Punjab, India
Chandigarh, India rajniajata@yahoo.com
Priyadarshi Ranjan
malhotrapankaj@hotmail.com
MS ( Surgery) MCh (Urology) PGI Chandigarh
Ravi Dara MD
Consultant Endourologist, Laparoscopic and
Paramjit Dhot Senior Resident
MD Post Doc Haematology AIIMS, FRSH (London), Kidney Transplant Surgeon
Medanta: The Medicity Hospital
MCHS (Toronto), FISHTM Chief, Division of Kidney Transplant Surgery
Gurgaon, Haryana, India
New Delhi Surgical Director, Incompatible Blood Type
rcdara@gmail.com
pamidhot@gmail.com Kidney Transplant
Fortis Hospital Ravisha Bhardwaj MBBS
Paramjit Kaur MD Mohali, Chandigarh, India Junior Resident
Assistant Professor priydarshiranjanurologist@gmail.com Department of Orthopedics
Department of Transfusion Medicine Government Medical College
Government Medical College and Hospital PS Ghalaut MD Amritsar, Punjab, India
Chandigarh, India Senior Professor and Head ravi_misha@yahoo.com
paramjit.gp71@yahoo.com Department of Medicine and Clinical
Hematology Ravneet Kaur  MD
Parveen Mittal MD Pt BD Sharma Postgraduate Institute of Professor and Head
Professor Medical Sciences (PGIMS) Department of Transfusion Medicine
Department of Pediatrics Rohtak, Haryana, India Government Medical College and Hospital
Government Medical College med.pgims@hry.nic.in Chandigarh, India
Patiala, Punjab, India rkbedi15@yahoo.com
doc_parveen@yahoo.co.in R Krishnamoorthy  MD
Associate Professor Reena Das  MD DNB
Department of Transfusion Medicine Professor
Poonam Singal  MD Department of Hematology
Pathologist Sri Ramachandra Medical College
and Hospitals Postgraduate Institute of Medical
Mata Kaushalya Hospital Education and Research
Patiala, Punjab, India Chennai, Tamil Nadu, India
rrkm29@yahoo.co.in Chandigarh, India
bansalrajesh1@gmail.com reenadaspgi@hotmail.com
Rajendra K Chaudhary  MD DNB
Prashant Agarwal  MD (Transfusion Medicine) Professor and Head
Rekha Hans  MD
PDCC (Apheresis and Component Therapy) Assistant Professor
Additional Professor Department of Transfusion Medicine
Department of Transfusion Medicine
Department of Transfusion Medicine Sanjay Gandhi Postgraduate Institute
Postgraduate Institute of Medical Education
Sanjay Gandhi Postgraduate Institute of of Medical Sciences (SGPGIMS)
and Research (PGIMER)
Medical Sciences (SGPGIMS) Lucknow, Uttar Pradesh, India
Chandigarh, India
Lucknow, Uttar Pradesh, India rkcchaud@sgpgi.ac.in
drhansrekha@gmail.com
prashantsgpgi@gmail.com
Rajesh B Sawant  MD Rimpreet Singh Walia  DNB
Consultant Consultant and Incharge
Prasun Bhattacharya  MD
Department of Transfusion Medicine Life Line Blood Centre
Assistant Professor
Hinduja Hospital Patiala, Punjab, India
Department of Immunohaematology and
Mumbai, Maharashtra, India rimpreet@yahoo.com
Blood Transfusion
sawantrb72@rediffmail.com
Medical College Hospital
RR Sharma  MD
Kolkata, West Bengal, India Rajesh C Gopal  MD Additional Professor
Visiting Consultant and Head of Department Former Technical Programme Manager Department of Transfusion Medicine
BM Birla Heart Research Centre Gujarat State AIDS Control Society Postgraduate Institute of Medical Education
Kolkata, West Bengal, India Ahmedabad, Gujarat, India and Research (PGIMER)
pbhattach@gmail.com dr_rajeshg@yahoo.com Chandigarh, India
rrsdoc@hotmail.com
Prathiba L Pujjita  MBBS Rajesh Kumar MD
Junior Resident Associate Professor RS Boparai MS
Vinayaka Missions Medical College Department of Transfusion Medicine Professor
and AMP Hospital Dayanand Medical College Department of Orthopedics
Salem, Tamil Nadu, India Ludhiana, Punjab, India Government Medical College
datlapoojith@gmail.com parul_pulkit@yahoo.co.in Amritsar, Punjab, India
x  Transfusion Update
Sandeep Kundra  MD PDCC (Neuro-anesthesia, Shruti Kakkar  MD Tarangini D MD
SGPGI, Lucknow) Fellowship in Comprehensive Department of Pediatrics
Associate Professor Hemato-oncology Institute For Child Health
Department of Anesthesia Assistant Professor Sir Ganga Ram Hospital
Dayanand Medical College and Hospital Department of Pediatrics Recipient Dr BC Roy Award
Ludhiana, Punjab, India Dayanand Medical College and Hospital Recipient Silver Jubilee Research Award
sandeepkundra07@gmail.com Ludhiana, Punjab, India New Delhi, India
drahujashruti@gmail.com
Sandeep Puri MD Tulika Chandra  MD
Professor and Head Sudhir Varma  MD DM Cardiology Associate Professor and Head
Department of Medicine Director and Principal Investigator Department of Transfusion Medicine
Dayanand Medical College and Hospital Sadbhavna Medical and Heart Institute King George Medical University
Ludhiana, Punjab, India Patiala, Punjab, India Lucknow, Uttar Pradesh, India
drsandeeppuri@yahoo.com drsudhirvarma@gmail.com drtulikachandra@gmail.com
Sangeeta Pathak MD Sunil D Khaparde  MD PhD
Senior Consultant and Head-Blood Bank Dengue Deputy Director General Umesh Sharma  MS MCH
Max Super Speciality Hospital DBTS Director of AIDS Control Consultant Hair Transplant
New Delhi, India New Delhi, India Laser and Cosmetic—Plastic Surgeon
sangeeta.pathak@maxhealthcare.com sdkhaparde.naco@gmail.com Paras Hospital
Haryana, India
Shanoo Mishra PhD Sunil K Arora  MD drumesh02@gmail.com
Programme Officer (QC) Professor
Directorate of AIDS Control Department of Immunopathology Varun Sharma MS
New Delhi, India Postgraduate Institute of Medical Education Senior Resident
poqc.naco@gmail.com and Research (PGIMER) Department of Kidney Transplantation and
Chandigarh, India Urology
Sharad Jain  MD arora.sunil@pgimer.edu.in Fortis Hospital
Associate Professor (Pathology) Mohali, Punjab, India
Netaji Subhash Chandra Bose Medical Suryatapa Saha
College and Hospital Junior Resident Vimarsh Raina  MD
Jabalpur, Madhya Pradesh Vinayaka Missions Medical College Director
and Deputy Registrar and AMP Hospital Lab and Transfusion Services
MP Medical Science University Salem, Tamil Nadu, India Medanta: The Medicity Hopsital
Jabalpur, Madhya Pradesh, India Gurgaon, Haryana, India
sharadjain@gmail.com Swati Kulkarni PhD
rainavimarsh@gmail.com
Scientist C
Sheetal Malhotra MD National Institute of
Assistant Professor Immunohaematology (ICMR)
Yazdi Italia  PhD
Ex-Hon Director
Department of Transfusion Medicine KEM Hospital Campus
Sickle Cell Anemia Control Program
Gian Sagar Medical College Mumbai, Maharashtra, India
A Go-NGO Partnership Program
Patiala, Punjab, India swatiskulkarni@gmail.com
Department of Health and Family Welfare
sheetalpgi2007@yahoo.com
Tanvi Sood Government of Gujarat
Shobini Rajan MD Demonstrator Pathology Hon. Secretary
Assistant Director General Government Medical College and Valsad Raktdan Kendra
National AIDS Control Organization Hospital (GMCH) Valsad, Gujarat, India
Ministry of Health and Family Welfare Chandigarh, India italialabvalsad@gmail.com
Director
National Blood Transfusion Council
New Delhi, India
shobininaco@gmail.com
shobininaco1@gmail.com 
Advisor Dr KK Talwar
Government of Punjab MD DM FAMS FNA FACC
Health and Medical Education FlMSA FIACS (Canada) DSc (hc)
Member, Punjab Governance Reforms Commission

Former Chairman, Board of Governors

Medical Council of India
Former President
National Academy of Medical Sciences
Former Director, PGIMER, Chandigarh
Former Professor and Head
Dept. of Cardiology, AIIMS, New Delhi

Foreword
It is a great pleasure to write a foreword for Transfusion Update. This is a result of the concentrated efforts
of the authors and the editorial team in bringing out a book containing the latest practices in transfusion
medicine. While covering a majority of the important and challenging topics in the field of transfusion, the
authors and editors have made sincere efforts to bring forth the practical points concerning patient care
from the transfusion medicine. The challenging problems seen in the practice of transfusion medicine are
dealt with in a simplified manner.
Transfusion Update is indispensable for transfusion medicine specialists and clinicians. I am sure
that postgraduate students will also find this book containing 81 chapters with pearls of immense value.
The book is having four sections consisting of clinical hemotherapy, cellular therapies, blood safety with pathogen reduction
technology and miscellaneous. To sum-up, the book is a practical and reference manual for clinicians on issues related to
transfusion medicine.
As this transfusion medicine update is being released on the occasion of the 39th Annual National Conference of the Indian
Society of Blood Transfusion and Immunohaematology (ISBTI) scheduled to be held at Government Medical College, Patiala,
Punjab, India, I congratulate Dr Kanchan Bhardwaj, Editor-in-Chief, for taking great pains in bringing out this volume of over
300 pages for the benefit of dedicated and busy clinicians of our country. I hope the TRANSCON 2014 and the book will add
to everyone’s knowledge and help everyone to face the challenges of transfusion practice in the years ahead. Dr BL Bhardwaj,
Dr Kusum K Thakur and Dr Rajni Bassi deserve special congratulations for successfully completing this important project.

(KK Talwar)

Punjab Governance Reforms Commission, Academic Block, Mahatma Gandhi State

Institute of Public Administrations, Institutional Area, Sector-26, Chandigarh 160019
E-mail: kktalwar@hotmail.com Fax: 91-172-2795121
 Foreword
It gives me immense pleasure in writing the foreword for Transfusion Update. At the outset,
I congratulate Dr Kanchan Bhardwaj, a simple, dynamic and competent academician who took this
tedious challenging job and came out with the book within short period of time which was the need of
the hour.
Publication of the book is unique in itself because it is for the first time in the history of ISBTI for more
than forty years of its inception that a publication of ISBTI in the form of the book has been launched and
the credit for this goes to Dr Kanchan Bhardwaj.
Blood transfusion services play a vital role in the modern health care system of both developed and
developing countries. Importance of transfusion medicine as a branch of medical science can never be undermined and is
now considered as an applied para-clinical discipline which demands a significant extent of academic knowledge. In a vastly
populated developing country like ours, blood transfusion services remain relatively inadequate, both in quantity and quality
of service. In this era of technology, although there is a lot of advancement in the medical field; but, unfortunately, the field of
blood transfusion has not kept pace with these advancements.
To improve blood transfusion services in our country, there is a genuine need of good textbooks and the endeavor
undertaken by Dr Kanchan Bhardwaj is a step towards this side. The book contains all the relevant chapters including more
recent topics on Cellular Therapies including Cord Blood Banking and Transplantation which shall be beneficial for all those
engaged in the field of transfusion medicine.

 TR Raina  MD (Path)
Former Professor and Head
Department of Transfusion Medicine
Government Medical College
Jammu, Jammu and Kashmir
Secretary General
Indian Society of Blood Transfusion and Immunohaematology (ISBTI)
Panchkula, Haryana, India
 Foreword
Transfusion medicine is a continually involving field. It has a wide spectrum of activities involving
the community for purposes of donor motivation, recruitment and retention, complex laboratory
technologies for blood component preparation and blood testing, regulatory aspects of blood transfusion
services and most importantly clinical transfusion practices, monoclonal antibodies for therapeutics and
cellular therapies.
Transfusion Update being released on the occasion of TRANSCON 2014 has been diligently compiled
by the Organizing Committee, under the initiative of Dr Kanchan Bhardwaj, Organizing Secretary of the
Conference. The contents fit in well with the theme—Optimizing Transfusion Therapy. Best practices,
recent advances and challenges in clinical transfusion in the fields of medicine, surgery, oncology, obstetrics and neonatology
have been included in the scientific program. Articles on scope of apheresis technology, special transfusion support in
hemoglobin disorders and hemophilia have also been contributed by experts.
A major section has been devoted to cellular therapies—the most challenging and controversial areas in regenerative
medicine. The role of platelets as growth and repair promoters, current status of gene therapy, blood grouping and cord blood
transplantation have also been included in the contents. The section on blood safety brings into focus the regulatory, ethical
and legal issues in transfusion medicine. It also highlights the hazards of blood transfusion–hemovigilance and its reporting
mechanism in the country. State-of-the-art technology, platforms for immunohematology, transfusion transmitted viral
infections and bacterial contamination of blood components have also been contributed by the experts. The contents also
included quality assurance principles and practices in transfusion services.
All the contributors deserve appreciation for the efforts, they have made in preparing manuscripts and I wish to put on
record my sincere praise and appreciation for the Organizing Committee for compiling updated scientific knowledge in the
field of transfusion medicine. Transfusion Update would definitely benefit all the readers and help in improving transfusion
practices.

Neelam Marwaha  MD FAMS FISHTM

Head
Department of Transfusion Medicine
Postgraduate Institute of Medical Education and Research (PGIMER)
Chandigarh, India
 Preface
The medical science is changing so fast that it becomes essential for transfusion medicine to keep a pace with the changes. The
transfusion therapy is the backbone of clinical practice and its outcome is a measure of patient care. The objective of this book
is to raise the level of our expertise in transfusion technology to latest international standards of safe blood transfusion and to
keep abreast with the latest innovations in the field of blood transfusion.
It is a pleasure to present Tranfusion Update to our worthy fellow transfusion specialists, and clinicians across the country.
The challenging problems seen in practice of transfusion medicine are dealt with in a crystal clear manner. The book will be an
indispensible tool in the transfusion medicine specialist and clinician’s kit. I am sure the postgraduates will also find this book
containing 81 chapters with particular focus on transfusion therapy very useful.
The topics covered under Clinical Hemotherapy are Transfusion Practice in Clinical Medicine, Transfusion in Surgical
Practice, Maternal, Fetal and Neonatal Transfusion Practice; Massive Transfusion; Platelet Therapy, Transfusion Practice
in Chronically Transfused Patients, Apheresis, Intervention in Bleeding Patients and Transfusion Practice in Oncology. The
section on Cellular Therapies has Stem Cell Therapy in Cardiovascular Diseases, Peripheral Artery Disease, Diabetes Mellitus,
etc. Blood Safety has NAT, Pathogen reduction technology, leucoreduction, etc. and Miscellaneous has Evidence-based
Transfusion Medicine, Blood Bank Accreditation, Molecular Genotyping and its Applications in Transfusion Practice, Rare
Blood Group Registry, etc. Details of latest topics will stimulate teachers of transfusion medicine and clinical specialists to do
more research in these fields.
The book is being released on the occasion of the 39th Annual Conference of Indian Society of Blood Transfusion and
Immunohaematology (ISBTI) being held at Government Medical College, Patiala, Punjab, India. I am sure that Transfusion
Update will add to everyone’s knowledge and armamentarium to face the challenges of transfusion practice in the years ahead.
I hope the readers will find the book useful for updating their knowledge regarding transfusion practice eventually
benefitting their patients.

 Kanchan Bhardwaj  MD MAMS FIAC FIMSA

Professor and Head
Department of Transfusion Medicine
Government Medical College
Patiala, Punjab, India
 Acknowledgments
For the first time in history of Indian Society of Blood Transfusion and Immunohaematology (ISBTI) that scientific lectures
have been compiled in the form of Transfusion Update which will be very useful for postgraduate students and teachers of
transfusion medicine, and all clinical specialties during their day-to-day practice. The idea was proposed in governing body
meeting of ISBTI at Patiala, Punjab, India, and was appreciated by all present especially Dr Sangeeta Pathak, Treasurer,
Indian Society of Blood Transfusion and Immunohaematology (ISBTI), Dr Ravneet Kaur, Editor, Asian Journal of Transfusion
Science (AJTS) and Professor and Head, Department of Transfusion Medicine, Government Medical College and Hospital,
Chandigarh, India and Shri M Satish, Vice President-Technical, ISBTI.
The first and the foremost heartfelt thanks to Dr Yudhbir Singh, National President, ISBTI, whose positive attitude has made
this venture a reality. I want to sincerely thank Dr KK Talwar, Advisor to Government of Punjab whose encouraging words
has gone a long way in pursuing this venture. I convey my special thanks to Dr TR Raina, Secretary General, ISBTI, who has
encouraged me to accomplish this task. I should not forget to pay my gratitude to Dr Neelam Marwaha, Professor and Head,
Department of Transfusion Medicine, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh,
India, for her consistent guidance. Without consistent perseverance of Dr BL Bhardwaj, this uphill task was not possible.
I express my gratitude and thanks to Dr KD Singh, Principal, Government Medical College, Patiala, Punjab, India, for
his kind and expert guidance. I am also thankful to all the clinicians of my institute, who have expressed the need for some
written guidelines about blood transfusion, which can be helpful in their day-to-day clinical practice and can guide the junior
residents about how best to utilize the Blood Transfusion Services (BTS) for safety of patients. I convey my special thanks to
Dr HS Sandhu (Medicine); Dr VK Sharda (Surgery); Dr Surinder Singh (Surgery), Former Principal, Government Medical College,
Patiala, Punjab, India; Dr HK Rao (Medicine), Former Head (Medicine); Dr Manjit Kaur Mohi; Dr Paramjit Kaur, Dr Khushpreet
Kaur and Dr Preet Kamal Sibia (Gynaecology); Dr JS Bhopal (Anesthesia); Dr Manjit Singh and Dr JPS Walia (Orthopedics),
Dr Mohinder Singh and Dr DP Singh (Surgery), Dr Ardaman Singh and Dr Naresh Garg (Medicine), Dr Manpreet Kaur and
Dr Meena Garg (Gynecology).
I highly appreciate guidance given by my seniors and colleagues from other institutes such as Dr Kulbir Kaur, Principal,
Punjab Institute of Medical Sciences, Jalandhar, Punjab; Dr RR Sharma, Additional Professor, Department of Transfusion
Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh; Dr RN Maharishi, Professor and Head,
Department of Transfusion Medicine, Guru Gobind Singh Medical College, Faridkot, Punjab; Dr Neeraj Sharma, Professor and
Head, Department of Transfusion Medicine, Government Medical College, Amritsar, Punjab; Dr Hitish Narang, Consultant,
Satguru Pratap Singh (SPS) Apollo Hospitals, Ludhiana, Punjab; Dr PK Sehgal, Head, Department of Transfusion Medicine,
Pt BD Sharma University of Health Sciences, Rohtak, Haryana, India; Dr Amarjit Kaur, Professor and Head, Department
of Transfusion Medicine, Dayanand Medical College, Ludhiana, Punjab; Dr Anil Vij, Professor and Head, Department of
Medicine, Institute of Medical Sciences, Jalandhar, Punjab; Dr Dharam Paul, Civil Surgeon (Retd); Dr ML Bansal, Editor-in-
Chief of Current Medical Journal of North Zone; Dr BK Aggrawal, Professor and Head, Department of Medicine, Maharishi
Markandeshwar Institute of Medical Sciences and Research, Mullana, Haryana; Dr RP Kudiar, Principal, Acharaya Shri
Chander College of Medical Sciences (ASCOM), Jammu, Jammu and Kashmir, India; Dr Anil Kumar Gupta, Professor and
Head, Department of Medicine, Acharaya Shri Chander College of Medical Sciences (ASCOM), Jammu, Jammu and Kashmir;
Dr PS Gehlot, Professor and Head, Department of Medicine, Pt BD Sharma University of Health Sciences, Rohtak, Haryana,
India; Dr Lovedeep Saini, Dr Sumeet Pal Saini, Dr Sukhwinder Singh, Joint Director, BS and QA, Punjab State AIDS Control
Society, Chandigarh; Dr Sartaj Kaur Dhillon and Dr Satwant Singh Dhillon, Wisconsin, USA; Dr AS Grover, Principal, Gian
Sagar Medical College and Hospital, Banur, Punjab, India.
I am thankful to all my friends and colleagues, who have helped me in writing the book in one way or the other. My
special thanks to Dr Gurdeep Kaur Bedi, Professor and Head, Biochemistry; Dr Shashi Prabha, Former Head, Department
of Transfusion Medicine; Dr MS Bal, Professor and Head, Department of Pathology; Dr Vijay Kumar Bodal, Dr Anil Suri,
Dr Mohanveer Kaur, Dr Maninder Kaur, Associate Professor, Biochemistry; Dr Avnish Kumar, Professor Physiology; Dr Usha
Chhabra, Professor and Head (Anatomy); Dr Subhash Kaushal, Professor, Department of Anatomy; Dr Anita Gupta, Professor
and Head, Pharmacology; Dr KK Aggarwal, Professor, Forensic Medicine; Dr DS Bhullar, Assistant Professor, Department
of Forensic Medicine; Dr Aradhana Sharma, Dr Poonam Singhal, Mr Mahesh Kumar, Mr Sukhvinder Singh and Mr Pawan
Kumar, Sandeep Kumari who have contributed in one way or the other for this manuscript. I express my special thanks to
Dr Manmohan Singh, Former President, Punjab Medical Council and Dr Sudhir Verma for their ever available guidance and
help.
xx  Transfusion Update
The book has emerged in its present form due to the untiring efforts of my Associate Editors—Dr BL Bhardwaj, Dr Kusum K
Thakur and Dr Rajni Bassi, and Assistant Editor—Dr Harnoor Singh Bhardwaj. My sincere thanks to the eminent contributors
for writing their chapters despite their own priorities.
I remember Dr Amrendra S Miranpuri, Assistant Professor, Neurosurgery Cerebrovascular, Stroke and Endovascular
Surgical, Co-Director, Neuromedicine Critical Care University of Rochester Medical Center, Rochester, New York;
Dr Guwatan Singh Miranpuri, Department of Neurosurgery, University of Wisconsin, Madison, USA; Dr Mandeep Batish;
Dr Aastha Miranpuri, Dr Ravisha Bhardwaj, Dr Jatinder Kumar, Mrs Ranju Saini, Er Aasheem Saini, Mr Rasheem Saini,
Mrs Nisha Bhardwaj, Dr Kavita Bhardwaj, Er Navpreet Singh, Er Shivani Bhardwaj, Er Ankush Bhardwaj, Dr Paritev Singh and
Mr Navnik Singh for their unstinting support, constant cooperation and prompt help.
My sincere thanks to Shri Jitendar P Vij (Chairman), Mr Ankit Vij (Managing Director), Mr Tarun Duneja (Director-
Publishing) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, deserve due credit for their personal
involvement in the excellent printing and publication of this book in a record time.

Editor-in-Chief
 Contents
Section 1: Clinical Hemotherapy
Transfusion Practice in Medicine
1. Transfusion in Bone Marrow Failure Syndromes 3
PS Ghalaut, Jitendra Kumar Pehalajani, Arvind Chahal
• Component Therapy  3
• Indications for RBC Transfusion  3
• Indications of Platelet Transfusion  4
• Granulocyte Transfusion  4
• Synthetic Oxygen Carriers  5
2. Transfusion Therapy in Autoimmune
Hemolytic Anemia 7
Rajesh B Sawant
• Indications for Transfusion  7
• Responsibilities of Transfusion Medicine  7
• Patient Management   8
• Autoimmune Hemolytic Anemia  8
3. Sugar Free Antibodies: A New Treatment for
Idiopathic Thrombocytopenic Purpura and
Autoimmune Hemolytic Anemia 9
HS Sandhu, Ishwar Chouhan
• Sugar Free Antibodies in Immune Thrombocytopenic
Purpura  9
• Sugar Free Antibodies in Autoimmune Hemolytic
Anemia  9
• Sugar Free Antibodies in Other Autoimmune
Diseases  11
• Future  11
4. Transfusion Practice in Disseminated
Intravascular Coagulation 13
Hitish Narang
• Pathophysiology  13
• Diagnosis  13
• Treatment  13
• Prognosis  14
5. Transfusion Practice in Oncology 15
Devinder Singh Sandhu
• Anemia  15
• Thrombocytopenia  16
• Neutropenia  18
6. Transfusion Practice in Coronary Interventions 20
Sudhir Varma
• Why Bleeding and Excess Morality Post Percutaneous
Coronary Intervention?  20
• Can Blood Transfusion in Percutaneous Coronary
Intervention Patients be Harmful?  21
7. Neurological ICU: Role of Red Blood Cell
Transfusion 24
BL Bhardwaj, Dharam Paul, Arun Bansal
• Role of Red Blood Cell Transfusions  24
• Adverse Effects of Transfusion  25
• Transfusion in the Neuro-ICU  25
8. Blood Transfusion Alternatives 30
Harnoor Singh Bhardwaj, BL Bhardwaj, Aastha Miranpuri
• Pharmacological Agents  30
• Recombinant Hematopoietic Growth Factors   31
• Red Cell Substitutes   31
• Platelet Substitute  33
9. Role of Whole Blood in Transfusion Practice 35
Ashok Sharma
• Indications of Whole Blood Transfusion  35
Transfusion Practice in Surgery
10. Management of Preoperative Anemia in Patients
Undergoing Elective Surgery 37
Manisha Shrivastava
• Prevalence Depends on Many Factors  37
• Recommendations for Detection, Evaluation and
Management of Preoperative Anemia in Orthopedic
Surgery [Network for Advancement of Transfusion
Alternatives (NATA)]  38
11. Blood Conservation Strategies 40
DP Singh
• Methods  40
12. Thromboelastography 42
Anupam Verma
13. Transfusion Support in Cardiac Surgery 44
Rajendra K Chaudhary
• Transfusion Variability in Cardiac Surgery  44
• Potential Benefits of Red Blood Cell Transfusion
in Cardiac Surgery  44
• Predictors of Allogeneic Red Blood Cell Transfusion
during Cardiac Surgery  45
14. Transfusion Practice in Arthroplasty 48
Manuj Wadhwa, Kunal R Patel
• Recent Studies and Recommendations for Specific
Orthopedic Problems  48
15. Restricting Transfusion Requirements in Major
Orthopedic Surgery and its Alternatives 51
Ravisha Bhardwaj, RS Boparai
• Techniques Applicable to Orthopedic Surgery  52
• Orthopedic Trauma  53
• Future  53
xxii  Transfusion Update
16. Transfusion Support in Neurosurgery 55 • Use of Ivig  84
Sandeep Kundra • Accommodation  84
• Threshold for Transfusion in Neurosurgery is • Current Protocol of Aboi-Kt  84
Uncertain  55 • Minimize Immunosuppression  84
• Blood Conservation Strategies  57 • Histological Findings in Aboi-Kt  84
• Incidence of Acute Cellular and Antibody Mediated
Maternal, Fetal and Neonatal Transfusion Practice Rejection in ABOi-KT  85
17. Noninvasive Prenatal Screening for Rh • Adverse Effect of ABOi-KT  85
Hemolytic Disease 60 24. Transfusion Practice in Heart Transplant 87
Manjit Kaur Mohi Hari Krishan Dhawan
• Possible Sources of Error  61 • Pretransplant Work-up  87
• Ethical Aspects   61 • Provision of Leukoreduced Blood Components  87
• Cost/Availability  61 • Transfusion Requirements during Transplant  87
18. Antenatal Antibody Screening and Intrauterine • Follow-up of Transplant Patients  87
• Role of Photopheresis and Therapeutic Plasma
Transfusion in Pregnancy 62
Exchange in Cardiac Transplant Patients  88
Amit Aggarwal
• Antenatal Testing and HDN Status in 25. Transfusion Practice in Liver Transplantation 89
Developing Countries  62 A Surekha Devi
• Intrauterine Transfusion  63 • Transfusion Predictors  89
19. Antenatal Screening of Thalassemia 65 • Drugs that Minimize Blood Loss  91
Reena Das • OLT without Transfusion  92
• Transfusion Support  92
20. Neonatal Transfusion Practice 67
A Surekha Devi Massive Transfusion
• General Recommendations (Fetuses,
Neonates, Infants and Children)  67 26. Massive Transfusion Protocol 95
• Transfusions of Preterm Infants  68 Kanchan Bhardwaj, Rajni Bassi
• Neonatal Transfusions  69 • Rationale  95
• Transfusions in Necrotizing Enterocolitis (NEC)  70 • Definition of Massive Transfusion  95
• Transfusions in Extracorporeal Membrane • Physiology of Hemorrhagic Shock   95
Oxygenation (ECMO)  70 • Coagulopathy of Hemorrhage and Massive
Transfusion   96
Transfusion Practice in Transplantation • Predicting Need for Mtp  96
21. Transfusion Practice in Bone Marrow • Adjuncts to Mtp  96
Transplantation and HLA Matching 71 • Hypothermia  96
M Joseph John • Rewarming Strategies  96
• Acidosis  98
• HLA Typing  71
• Tranexamic Acid   99
• Transfusion Practices  71
• Resuscitation in Patients Requiring Massive
• ABO Incompatibility in BMT: Graft Management  73
Transfusion  99
• Blood Groups used for Transfusion Support  74
• Thromboelastography (Teg) and Rotational
22. Transfusion Practice in Renal Transplants Thromboelastometry (Rotem)  99
Recipients 76 • Factor rviia and Mtp  99
Vimarsh Raina, Aseem K Tiwari, Ravi Dara • Complications of Mtp  99
• Auto-recognition  76 • Mtp after Control of Hemorrhage  100
• Allo-recognition  76 • Mtp in Pediatrics  100
• Allograft Rejection  76 27. Massive Transfusion in Obstetrical
• Evolution of Transfusion in Renal Transplantation   77 Hemorrhage 102
23. Incompatible Blood Type Kidney Sangeeta Pathak
Transplantation 81 • Principle  102
Varun Sharma, Priyadarshi Ranjan • Definition  102
• Abo Antigens and Antibodies  81 • Blood Component Therapy  103
• History  82 • Recombinant Factor VIIA  104
• Aboi-Kt Preoperative Management  82 • Adverse Effects of Massive Transfusion  104
 Contents  xxiii

Platelet Therapy 37. Therapeutic Erythrocytapheresis and

Leucapheresis 135
28. Inherited and Acquired Platelet Disorders: Nidhi Mehta
Diagnosis and Therapy 108 • Plasma Exchange (PE)  135
R Krishnamoorthy • Cytapheresis  135
• Methodology  108
• Discussion  108 38. Red Cell Exchange in Clinical Practice 137
Joshua Daniel Jeyakumar, Prakash H Muddegowda,
• Diagnosis  109
Jyothi B Lingegowda
• Management  109
• Red Blood Cell Exchange Technique  137
29. Transfusion Practice in Dengue 110 • Indications of Red Blood Cell Exchange  137
BL Bhardwaj, Harnoor Singh Bhardwaj • Sickle Cell Disease  137
• Clinical Management  110 • Malaria  138
• Babesiosis  138
30. Fetal and Neonatal Alloimmune • Nitrobenzene and Other Poisonings  138
Thrombocytopenia 116 • Hemolytic Disease of Newborn  138
Prasun Bhattacharya • Miscellaneous  138
• Pathogenesis of FNAIT  116
• Clinical Presentation  116
Chronically Transfused Patients
• Laboratory Investigations  116 39. Transfusion Practice in Thalassemia 140
• Therapy and Management  117 Gagandeep Kaur
• Newborns  117 • Pathophysiology  140
31. Clinicopathological Syndrome in Immune- • Transfusion Therapy  140
• Whom to Transfuse  140
mediated Heparin-induced Thrombocytopenia 118 • Compatibility Testing   140
P Arumugam • Transfusion Regimen  141
• Pathogenesis  118 • Transfusion Interval  141
• Clinical features  118 • Characteristics of Blood Products for
• Diagnosis  119 Transfusion  141
• Management  121 • Effectiveness of Transfusion Therapy  141
• Prevention  122 • Problem of Thalassemia Transfusion Programs is
• Information to the Patient and Record Keeping  122 Developing Countries  142
32. Single Donor Platelets Versus Random 40. Pediatrician and β-Thalassemia 143
Donor Platelets 123 Parveen Mittal
Rajni Bassi, Ajata Shatru Kapoor • Road Map  143
• Preparation of Platelet Concentrate  123 • Management  144
• Random Donor Platelets   123 • Complications of Thalassemia   144
• Follow-up  145
• Single Donor Platelets/Plateletpheresis  124
• Prognosis   145
33. Prophylactic Platelet Transfusion 126 • Prevention  145
Poonam Singal • Prenatal Diagnosis   145
• Transfusion Triggers  126 41. Iron Overload in Chronically Transfused
Patients: Diagnosis and Treatment 147
Apheresis Praveen C Sobti, Shruti Kakkar
34. Maximizing the Resources: Multicomponent • Assessment of Iron Overload  147
• Desferrioxamine  149
Collection 129
Prashant Agarwal 42. Transfusion in Sickle Cell Anemia 153
Yazdi Italia
35. Therapeutic Plateletpheresis 131
Tulika Chandra 43. Advanced Therapies for Treatment
• Therapeutic Plateletpheresis  131 of Hemophilia 155
Tarangini D, Anupam Sachdeva
36. Therapeutic Plasma Exchange 133 • Advanced Therapies for Hemophilia
Aseem K Tiwari under Trial  155
• Plasma Exchange as an Alternative to • Management of Inhibitors  156
Immunoglobulin  134 • Management of Hemophilic Arthropathy  156
xxiv  Transfusion Update
Intervention in Bleeding Patients
44. Bleeding Disorders: Lab Diagnosis 157
Mohanvir Kaur, Dinesh Ahluwalia
• General Aspects  157
• Laboratory Investigations  157
45. Management of Coagulopathy in
Liver Diseases 162
Meenu Bajpai
• Defects in Primary Hemostasis  162
• Defects in Secondary Hemostasis  162
• Overview of Management  163
46. Recombinant Activated Factor VII in
Clinical Practice 166
Ravneet Kaur, Kshitija Mittal, Tanvi Sood
• Mechanism of Action   166
• Indications   166
• Off-label Uses   166
47. Plasma Versus Recombinant Clotting Factors 168
Rajesh C Gopal
• Clotting Factor Concentrate  168
• Demerits of Plasma Factor Concentrates  168
48. Solvent Detergent Treated Plasma in
Clinical Use 171
M Joshua Daniel Jeyakumar, Suryatapa Saha, Prakash H
Muddegowda, Prathiba L Pujjita
• Production of SD Plasma  171
• Properties of SD Plasma  171
• Octaplas Follow-up Study  172
• Pharmacoeconomics  172
• Conclusion with European SD Plasma  172
49. Cryoprecipitate 174
Aradhna Sharma
• Cryoprecipitate Constituents and their • Contraindications  199
Stability  174
• Indications  174
• Common Misuses  174
• Doses and Administration of Factor VIII
Concentrates  174
• Preparation, Storage and Quality Control  175
• Pooling of Cryoprecipitate  176
• Potential Adverse Reactions  176
Section 2: Cellular Therapies
50. Stem Cell Transplantation: Brief Overview 179
Pankaj Malhotra
51. Stem Cell in Regenerative Medicine:
Applications and Cautions 181
RR Sharma
• Translation of Stem Cell Science into Regenerative
Medicine  181
• Threats to Clinical Translation and to the Integrity of
Regenerative Medicine  182
52. Stem Cell Therapy in Cardiovascular Diseases 184
Hamed Bashir, Ajay Bahl, KK Talwar
• Embryonic Stem Cell  184
• Adult Stem Cells  184
• Mechanism of Action of Stem Cells  184
• Types of Cells Contemplated for Cellular Regenerative
Therapy  184
• Methods of Stem Cell Delivery  185
• Stem Cell Therapy for Cardiac Repair  185
• Results of Stem Cell Transplant in Clinical Trials  186
• Concerns  187
53. Stem Cell Therapy for Diabetes Mellitus 189
Rajesh Rajput
• Stem Cell Therapy for Diabetes  190
• Ethical Issues with Use of Stem Cells  191
54. Stem Cell Therapy in Peripheral Artery Disease  193
Bimal Kumar Agrawal, Mini Bhatnagar Sud
• ABI and Diagnosis of PAD  193
• Stem Cell Therapy for PAD  194
• Clinical Trials on Stem Cell Therapy for PAD  195
• Current Status of Stem Cell Therapy in PAD  196
55. Role of Platelet Rich Plasma in Regenerative
Medicine 197
Kusum K Thakur, Alpna Thakur, Achhar Singh
• Historical Perspective  197
• Basic Science of Platelet Rich Plasma  197
• Platelet Rich Plasma Therapy  198
• Preparation of Platelet Rich Plasma and its
Application  198
• Therapeutic Platelet Products  199
• Indications  199
• Safety Profile  199
• Future of Platelet Rich Plasma in Regenerative
Medicine  200
56. Role of Platelet Rich Plasma in Plastic Surgery 202
Umesh Sharma
• Platelet Rich Plasma  202
• Rationale for Use of Platelet Rich Plasma  202
• Platelet Rich Plasma in Plastic Surgery   202
• Safety, Complications, Contraindications
to Use of Platelet Rich Plasma  203
57. Cord Blood Banking and Transplantation 204
Paramjit Dhot
58. Erythropoiesis Stimulating Agents versus
Red Cell Transfusion 206
Sandeep Puri
• Erythropoiesis Stimulating Agents  206
• Red Cell Transfusion  208
 Contents  xxv

59. Natural Killer Cells in Tumor and • Direct Methods of Bacterial Detection  238
Transplantation 211 • Direct Rapid Detection Methods   239
Sunil K Arora, Rajendra Kumar • Pathogen Reduction  239
• Origin and Distribution of Natural Killer Cells  211 66. Pathogen Reduction Technology 242
• Mechanism of Cytotoxicity  211 Kanchan Bhardwaj
• Role of Natural Killer Cells in Transplant • Pathogen Inactivation in Platelet
Immunology  212 Concentrates  242
• Natural Killer Cells and Cancer Surveillance  212 • Platelet Concentrates with Amotosalen
60. Potential and Hazards of Gene Therapy 214 Photochemical Treatment   242
Ajit C Gorakshakar • Riboflavin and Ultraviolet Light Treated
Platelets  243
• Historical Aspect  214
• Methylene Blue Treated Platelets  243
• Gene Therapy in Transfusion Medicine   214
• Pathogen Inactivation in Fresh Frozen Plasma
• Prerequisite for Gene Therapy   215
Preparation  243
• Gene Delivery Systems  215 • Potential System for Pathogen Inactivation
• Vector Design   215 in Red Cell Concentrates  243
• Viral Delivery Systems   215
• Nonviral Delivery Systems   215 67. Discarding of Blood and Blood Components 245
• Regulatory Issues  216 Bharat Singh, Preeti Dewakar
• Recommendations  251
Section 3: Blood Safety 68. Adverse Transfusion Reactions 252
Manoj A Kahar
61. Leucoreduction 221 • Classification  252
Kanchan Bhardwaj • Acute Adverse Transfusion Reaction
• Approximate Leucocytes in Various Red Blood with Immune Causes  252
Cell Preparations  221 • Investigations to be Done for Immediate
• Clinical Indications for Leucoreduction  221 Hemolytic Transfusion Reaction  254
• Methods of Leucodepletion  222 • Acute Adverse Transfusion Reaction
• Adverse Effects of Filtration and with Nonimmune Causes  256
Leucodepletion  224 • Delayed Adverse Transfusion Reaction
• Universal Leucodepletion versus Selective with Immune Causes  257
Leucodepletion   224 • Delayed Adverse Transfusion Reaction
• Leucodepletion—Indian Perspective  225 without Immune Causes  258
62. Improving Blood Donor Screening by Nucleic 69. Hemovigilance and its Launch in India 260
Acid Technology 226 Neelam Marwaha
Rekha Hans • Hemovigilance Systems  260
• Need for Nucleic Acid Technology  226 • Outcome of Hemovigilance around the World  260
• Nucleic Acid Technology  226 70. Advances in Serological Assays for Screening
• Blood Donor Screening  226 Blood Donors for Infectious Agents 264
• Experiences of Different Countries  227 Paramjit Kaur
63. ID-NAT Versus MP-NAT 230
Rajesh Kumar Section 4: Miscellaneous
64. Occult Hbv Infection: A Risk for Transfusion 232
Sheetal Malhotra 71. The Evidence-based Transfusion Medicine 269
Kulbir Kaur
65. Current Status of Bacterial Detection in • Evidence-based Red Blood Cells Utilization
Blood Components 236 Guidelines  270
Mina Sidhu • Evidence-based Platelet Utilization Guidelines  270
• Clinical Relevance  237 • Evidence-based Fresh Frozen Plasma Utilization
• Methods to Reduce Risk of Post-transfusion Guidelines  271
Sepsis  237 • Evidence-based Cryoprecipitate Utilization
• Methods to Detect Bacterial Contamination Guidelines  271
in Blood Component   237 • Limitations of Evidence-based Transfusion
• Indirect Rapid Bacterial Detection Tests  237 Medicine  271
xxvi  Transfusion Update
72. Blood Bank Accreditation 273
BK Rana
• Types of Blood Banks/Centres  273
• Scope of Accreditation  273
73. National Plasma Policy for Access to Plasma
Derived Proteins for Clinical/Therapeutic Use 278
Harprit Singh, Shobini Rajan, Shanoo Mishra,
Sunil D Khaparde
• Objectives of the Policy  278
74. Ethical Issues in Transfusion Medicine 281
Kusum K Thakur, Achhar Singh
• International Society of Blood Transfusion Code • Quality Control in Reagent Red Cells  294
of Ethics  281
• A Code of Ethics for Blood Donation and
Transfusion  281
• Ethical Issues Related to Donors  282
• Ethical Issues Related to Patients  283
• Ethical Issues Related to Blood • International Rare Donor Program   297
Establishments  283
• Future Prospectives   283
75. Gel Based Technology: Past, Present and Future 285
Nidhi Bhatnagar
• Principle of Gel Technology  285
• Practical Applications of Gel Technology  286
• Advantages of Gel Technology  286
• Past, Present and Future  287
76. Automated Blood Grouping and Antibody
Screening 288
Ashish Jain
• Automation in Serologic Testing  288
• Solid-phase Immunoassays  288
• Erythrocyte-magnetized Technology  289
• Validation of Automated Systems  291
• Edge of Automation  291
77. Molecular Genotyping and its Applications
to Transfusion Medicine 292
Swati Kulkarni
• Solving ABO Blood Group Discrepancies   292
• Identification of Weaker and Partial Variants
of RhD Gene   293
• RhD Zygosity Testing  293
• Identification of DEL Phenotypes   293
• Prenatal Diagnosis of RhD in Fetus   293
• Antigen Profiling in DAT Positive Cases  294
• Differentiation between Alloantibody and
Autoantibody  294
• Screening for Rare Blood Group Donors  294
78. Rare Blood Group Registry 297
Swati Kulkarni
• Defining Rare Blood   297
• National Status   298
• Need for National Registry   298
• Identification of Rare Donors   299
• Methods for Typing Blood Group Types   299
• Future Directions   299
79. New Versus Old Blood 301
Rimpreet Singh Walia
80. Importance of Safe Blood Administration
Practices 305
Sharad Jain
81. Strategies to Reduce Unnecessary
Transfusions 308
Hemchandra Pandey, Rajendra K Chaudhary
Index 311
 Section

1
Clinical
Hemotherapy
 Transfusion Practice in Medicine
Transfusion in Bone
Marrow Failure Syndromes
PS Ghalaut, Jitendra Kumar Pehalajani, Arvind Chahal
Introduction
Bone marrow failure syndromes may primarily affect a single
or multiple hematopoietic lineages, which may result in
decrease in one or more cell line in the blood. They can be
congenital or acquired.1
Congenital/Inherited Aplastic Anemia
1. Fanconi anemia (FA)
2. Dyskeratosis congenita (DC)
3. Shwachman-Diamond syndrome (SDS)
4. Congenital amegakaryocytic thrombocytopenia (CAMT)
5. Thrombocytopenia with absent radii
6. Kostmann’s syndrome
7. Congenital dyserythropoietic anemias.
Acquired Bone Marrow Failure
1. Acquired aplastic anemia
2. Idiopathic thrombocytopenic purpera
3. MDS
4. PNH
5. Megaloblastic Anemia
6. Hematological malignancy—Lymphoma, leukemia, mul-
tiple myeloma, myelodysplastic syndromes, myeloprolif-
erative syndromes.
7. Toxins and drugs.
Management of these etiologies are important. It is also
imperative to maintain the near normal blood counts by
blood transfusion. Appropriate and early transfusion can also
decreases mortality as well as morbidity of the patients. It
has been also proven useful to transfuse only the component
which is deficient. This article discusses the indications
of blood and blood products in the bone marrow failure
syndrome.
Component therapy
Component therapy is also called “Quiet revolution in Blood
Transfusion Therapy”.2
1
One unit of blood can be separated into:
(i) A red cell preparation containing all the red cells of a
unit of whole blood.
(ii) A platelet concentrates having approx. 70% of the
platelets.
(iii) Residual plasma is fractionated into
– Albumin
– Immunoglobulin
– Preparation of various clotting factors.
Indications for RBC Transfusion3
1. Hemoglobin < 7 g/dl in asymptomatic patients.
2. Hemoglobin < 10 g/dl in cases of increased risk of ischemia,
i.e. IHD, pulmonary disease.
3. Transfusion for a predetermined therapeutic program
such as bone marrow suppression, i.e. PNH, AA.
4. Symptomatic anemia resulting in:
– Tachycardia
– Mental states changes
– Angina/ECG changes of ischemia
– Shortness of breath, and dizziness on mild exertion.
Precautions
• Leuco-reduced packed red blood cells (PRBC) and platelets
should be used to reduce the risk of alloimmunization and
adverse reactions.
• It is important to avoid transfusion of blood from any
blood relative to prevent the risk of graft versus host-
disease (GvHD).
• The risk of CMV transfusion can be reduced by screening
by CMV antibodies or by using leuco-reduced blood
component.
• Red cell concentrates: Suspension of red cells prepared
by removing approx. 200 ml of plasma from whole blood.
Concentrated red cells and colloid or electrolyte are equally
effective. it is a very effective means to correct anemia. it
also reduces the risk of hypervolemia. Hematocrit of whole
blood is around 40%, in comparison to it hematocrit of red
4  Section 1  Clinical Hemotherapy
Table 1: Comparison between rcc and whole blood
RCC Whole blood
Total volume 300 500
Red cell volume 200 200
Plasma volume 100 300
Hematocrit 70 40
Albumin content 4 12
cell concentrate is around 70% (saline may be added to
decrease hematocrit).4 The various differences between
red cell concentrate and whole blood are as given in
Table 1.
• Packed red cells: In 90% of cases anemia is chronic and
requires red cells only.
Transfusion with packed red cell is twice as efficient as
whole blood in correcting anemia. Plasma can be saved for
use in others patients.
• Frozen thawed red cell: These can be done with
cryopreservation techniques. It is an expensive process.
Blood can be stored up to 10 years by such means.
Indications
• Provision of red blood cells to individuals with rare
blood groups.
• Autotransfusion to individuals with red cell alloantibody
which make the provision of compatible donor blood
difficult.
• Clinical situations with the need to minimize
alloimmunization in individuals undergoing chronic
transfusion therapy.
• Reconstituted thawed red cells not to be stored longer
than 24 hrs.
• Leukocyte depleted red cells5
– Indicated in severe febrile non hemolytic transfusion
reactions.
– Special filters are used along with micro aggregate filters
for removal of platelets, WBC and fibrin (40 micron).
Indications of Platelet Transfusion
Platelet transfusion is to be given to prevent bleeding due
to thrombocytopenia. In a bleeding patient a platelet count
above 50,000 µL should be maintained. In a surgical patient,
30,000–50,000 µL will be adequate for most surgeries but
for risk procedures a count of 100,000 µL is recommended.
Family donor or human leukocyte antigen (HLA) matched
platelets are indicated when patients have become refractory
to random donor platelet transfusion. Platelet transfusion
may be contraindicated in thrombotic thrombocytopenic
purpura (TTP) and in idiopathic thrombocytopenic purpura
(ITP) increments are usually poor.6
Platelet Transfusion to Control Bleeding
• Aim of platelet transfusion is to balance the risk of
hemorrhage against the risk of repeated transfusion.
Table 2: Recommended coagulation parameters for common
procedures
Procedure Platelet count/µL INR
Lumbar puncture 50,000 1.5
Paracentesis 30,000 2.0
Thoracentesis 50,000 1.5
Transbronchial lung biopsy 50,000 1.5
Subclavian/IJV line 30,000 1.5
Renal biopsy 50,000 1.5
Liver biopsy 50,000 1.5
• Mucosal/retinal hemorrhage or purpura in a patient with
thrombocytopenia is an indication for platelet transfusion
repeated transfusion may be needed.
• Failure to control bleeding may be due to infection,
splenomegaly and antibodies against leucocytes/platelet
antigens.
• Frequent platelet transfusion and use of HLA-compatible
PC may help to control bleeding.
Platelet Transfusion to Prevent Bleeding
Platelet are not given for stable afebrile patients, if the
platelet count is above 10,000/µl. In febrile patients a higher
threshold of 20,000/µl is taken. In stable patients, platelet
transfusion should be given to maintain the platelet count
at the chosen level. Transfusion, every 2 or 3 days is usually
sufficient. As invasive procedures are to be done cautiously in
patients with coagulation disorders, recommended platelets
levels and INR for various procedures are given as in Table 2.
Platelet Concentrate
It can be in form of platelet rich plasma (PRP) or platelet
concentrate. PRP of same ABO blood type as of the patients
can be used only. Platelet concentrates are 15–20 ml per
unit. It requires 6–8 units of platelet concentrates/m2.
Platelets should be given intravenous in plastic syringes in
thrombocytopenia with bleeding. The average rise in platelet
count with relation to body weight and platelet transfusions
are given in Table 3.7
Granulocyte transfusion
Impact of Neutropenia
Severe bacterial and fungal infections in the setting of
prolonged chemotherapy-associated neutropenia continues
to be a significant source of morbidity and mortality in
the modern treatment of malignancy. The risk of serious
bacterial infection first appears as the neutrophil count falls
to <1.0 × 109/L and risk increases rapidly at <0.5 × 109/L. The
introduction of modern apheresis techniques, the use of
growth factors to increase donor granulocyte yields.8
 Chapter 1  Transfusion in Bone Marrow Failure Syndromes  5

Table 3: Expected platelet increment • CMV negative, immunosuppressed and HIV positive
patients are at high risk for CMV infection.
Weight 1 Unit 4 Units 6 Units
• Bacterial testing in platelet concentrates has significantly
(kg) 1.0 × 1011 4.0 × 1011 6.0 × 1011
decreased this risk from platelet transfusion.
23 kg 22,000 88,000 132,000
45 kg 11,000 45,000 66,000 Transfusion Reactions
68 kg 7,400 30,000 44,000
A transfusion should be stopped immediately whenever a
91 kg 5,500 22,000 33,000 transfusion reaction is suspected.
• Hemolytic transfusion reaction: Fatal hemolytic
transfusion occurs once in 60,000 transfusions. It usually
Indications of Granulocyte Transfusions9 occurs after transfusion of incompatible blood component.
Fever, hypotension, nausea, vomiting, tachycardia,
• Granulocyte therapy may warrant consideration in
dyspnea, chest or back pain and hemoglobinuria are various
severely neutropenia patients with bacterial infections
signs and symptoms. May cause renal failure and DIC.
that are unresponsive to typical antimicrobial therapy.
• Delayed hemolytic transfusion reaction: These reactions
• A brief trial of granulocyte infusions may be warranted in
occurs in previously sensitized patient and can cause
patients with self-limited neutropenia and documented
symptomatic or asymptomatic hemolysis several days
fungal infection who are refractory to standard antifungal
after transfusion.
therapy.
• Febrile transfusion reaction: These reactions are
• Although prophylactic granulocyte transfusion may
due to sensitization to antigens on cell components
decrease the risk of septicemia, the increased incidence of
particularly leukocytes. Leukocyte depletion and removal
adverse effects observed with this therapy may outweigh
of plasma may be helpful. Rarely may be due to bacterial
the beneficial effects.
contamination.
• Transfusion related acute lung injury: These reactions
Doses and Target Level occur when donor plasma contains antibodies against the
• The recommended dose is 1 × 109 granulocytes/kg of body patient’s HLA or leukocyte specific antigen. Symptoms
weight. include dyspnea, hypotension and fever usually 30
• Granulocytes should be administered on a daily basis until minutes to 6 hrs after transfusion. Ventilatory support may
the patient’s endogenous neutrophil count rises to 0.5 × be required.
109/L or until the infection clears. • Urticarial and allergic type reactions: Most commonly
due to allergies due to specific proteins in donor’s plasma.
If severe washed RBC’s should be used. IgA deficiency may
Synthetic oxygen carriers be thought in severe reactions.
Various oxygen carrier compound available including the
following: Conclusion
• Perfluorochemicals: These are fluorinated hydrocarbons,
Stored in frozen state Fluosol-DA has been found to Early diagnosis of the etiology and specific therapy is
decrease the size of myocardial and cerebral infarctions in imperative in bone marrow failure syndromes. Appropriate
animals. and timely transfusion can reduce mortality as well as
• Chemically modified hemoglobin solutions: They morbidity of the patients, thus the judicious use of the blood
are used to increase oxygen transport in place of viable product is needed to minimize the side effect. It is essential
red cells. They have been used to supply oxygen in to use blood component therapy and not the whole blood so
experimental animals. that not only there is no waste of other blood component but
also there are less complications and transfusion reactions.

Transfusion Related Risks

REFERENcES
Following infection can be transmitted.10
1. Young NS (Ed). Bone Marrow Failure Syndromes. Philadelphia,
The current estimated risks are WB Saunders. 2000;24:12-5.
• HIV <1:1,900,000 2. Mintz PD, Dzik WH. Transfusion. 2009;49(7):1282-5.
• Hepatitis C < 1:1,000,000 3. Liumbruno G. Recommendations for the transfusion of red
blood cells. Blood Transfus. 2009;7(1):49-64.
• Hepatitis B <1:1,000,000
4. Corwin HL, Carson JL. N Engl J Med. 2007;356(16):1667-9.
• HTLV I and II < 1:641,000
6  Section 1  Clinical Hemotherapy
5. Bordin JO, Heddle NM, Blajchman MA. Biologic effects of
leukocytes present in transfused cellular blood products.
Blood. 1994;84:1703-21.
6. Slichter SJ, et al. NEJM. 2010;362:600-13.
7. Stroncek DF, Rebulla P. Platelet transfusions. Lancet. 2007;
370(9585):427-38.
8. Darmon M, Azoulay E. Impact of neutropenia duration
on short-term mortality in neutropenic critically ill cancer
patients. Intensive Care Medicine. 2002;28:1775-80.
9. Seidel MG, Einsele H. Randomized phase III study of
granulocyte transfusions in neutropenic patients. Bone
Marrow Transplantation. 2008. p. 237.
10. Eleftherios C Vamvakas, Morris A. Blajchman transfusion and
the available strategies for their preventionTransfusion-related
mortality: the ongoing risks of allogeneic blood. 2009;113:
3406-17.
Transfusion Therapy in
Autoimmune Hemolytic Anemia
Rajesh B Sawant
Indications for Transfusion
Red blood cell transfusion is a significant component of the
supportive care for all patients with Autoimmune hemolytic
anemia (AIHA). Although many patients will present with mild
to moderate anemia that does not require urgent transfusion,
an occasional patient presents with life-threatening anemia,
and the need for red blood cell transfusions is emergent.
Patients with auto-antibodies requiring transfusion may
pose difficulties in compatibility testing, and may be at
risk for hemolyzing transfused blood and/or developing
alloantibodies.
Careful communication between the clinician and the
transfusion service is required, since transfusion may be
required before the serologic evaluation is completed. Even
after thorough serologic evaluation, the optimal blood for
transfusion may be serologically incompatible. The clinician
must understand that in some cases, serologically incompatible
blood is safe for transfusion and can be expected to have in vivo
survival comparable to the patient’s own red cells. Reluctance
to transfuse these patients because of serologic incompatibility
or an incomplete work-up can be devastating. Patients with
severe anemia may appear to be hemodynamically stable,
but these patients have life-threatening anemia and should
be transfused immediately regardless of the status of the
serologic evaluation or compatibility.
Responsibilities of
Transfusion Medicine
Clinical
• Advise the clinician(s) of the duration of delay in obtaining
compatible blood or discuss transfer of the patient to a
larger center.
• Be familiar with the policies, processes, and procedures to
release “least incompatible” blood.
2
• If the case is complex and the transfusion services cannot
readily perform the appropriate investigations or provide
appropriate blood, then the transfusion medicine-MD
should consult a hematologist.
Technical
• Investigations should be expedited to obtain crossmatch
compatible blood as quickly as possible.
• Perform appropriate technical procedures.
• If the transfusion service is unable to perform these
investigations, send samples immediately to a reference
regional laboratory for investigation.
• Consult with seniors as indicated in technical procedures,
or as necessary based on technologist’s skills, additional
information on the clinical situation, or laboratory
examination results.
Pretransfusion Examinations
The following examinations need to be done on pre-
transfusion samples:
• Preliminary antibody investigation to determine whether
the auto-antibody demonstrates any specificity.
• Other examinations to identify underlying alloantibodies.
Such as prewarm, autoadsorption, differential alloadsorp­
tion.
• Full red blood cell phenotype may be required in some
cases.
• Red cells provided for transfusion should not express
clinically significant antigens which the patient lacks, or
should be negative for clinically significant antigens, if the
patient phenotype is unknown.
Selection of Blood for Transfusion
Transfusion management is complicated for patients with
AIHA, due to their serologic complexities. Although patients
8  Section 1  Clinical Hemotherapy
with AIHA can present with an ABO discrepancy, ABO
typing can usually be performed following removal of IgG
autoantibody in warm AIHA or warm washing red cells to
remove IgM autoantibody in cold agglutinin syndrome. In
an emergency or if results are not clear-cut, Group O donor
red cells can be used. Rh typing can also be difficult if the
patient’s cells are heavily coated with autoantibody. Low
protein, monoclonal reagents are available for Rh typing in
the setting of immunoglobulin-coated red cells.
The most pressing problem in a patient with previous
pregnancy or transfusions is detecting and identifying
alloantibody, which may be hidden or masked by the
autoantibody. The exclusion of underlying clinically
significant allo-antibodies is time consuming, and the clinical
situation may not allow for completion of these studies
before transfusion is needed. In the untransfused patient,
autologous adsorption studies are required to investigate
the presence of underlying alloantibody. If the patient has
been transfused in the preceding 3 months, more complex,
differential allogeneic adsorption studies are required.
In the un-transfused patient, determination of the red
cell phenotype is invaluable. Techniques and procedures
are available to dissociate autoantibody so that phenotyping
can be performed. Once a red cell phenotype has been
determined, this phenotype can guide the exclusion of allo-
antibodies by indicating which antigen specificities are
at risk of eliciting an alloantibody. Shirey et al (2002) has
shown that when available, phenotypically matched red
cells are an efficient method for providing safe red cells for
transfusion in this setting. Additionally, patients with AIHA
are at risk for requiring chronic transfusion therapy; knowing
the red cell phenotype of the patient is helpful in identifying
alloantibodies if they develop over time.
Patient Management
• In a stable patient, hemoglobin levels as low as 60 g/L may
be tolerated in an attempt to avoid exposing the patient to
blood transfusion, and the attendant risk of alloantibody
development.
• In a symptomatic patient, blood transfusion should not be
withheld and can be initiated under a steroid cover.
• Red cells for transfusion should be selected that are
the “least incompatible”, now preferably termed “Best
Matched” with the patient’s plasma/serum to minimize
the risk of alloantibody formation.
Role of Steroids in Treatment of AIHA
The mainstay of treatment of newly diagnosed primary
warm antibody autoimmune hemolytic anemia (WAIHA) is
glucocorticoids (steroids). According to accepted recommen­
dations, start treatment immediately with an initial dose
of 1 mg/kg/d prednisone orally or methylprednisolone
intravenously. This initial dose is administered until a
hematocrit of greater than 30% or a hemoglobin level greater
than 10 g/dL is reached. Once the treatment goal is achieved,
the dose of prednisone is reduced to 20–30 mg/d within
a few weeks. Thereafter, the prednisone dose is tapered
slowly (by 2.5–5 mg/d per month) under careful monitoring
of hemoglobin and reticulocyte counts. An alternate-day
regimen (reducing the dose gradually to nil on alternate days)
may reduce the side effects of steroids. If the patient is still in
remission after 3–4 months at a dose of 5 mg of prednisone/
day, an attempt to withdraw steroids is made. If a patient is
refractory to the initial corticosteroid treatment, a diagnostic
re-evaluation with regard to a possible underlying disease
should be made. Patients with malignant tumors, benign
ovarian teratomas, or with warm IgM antibodies are often
steroid-refractory.
Autoimmune Hemolytic Anemia
Controversies Regarding Transfusing
Crossmatch-Incompatible Blood
In patients with autoimmune hemolytic anemia (AIHA),
antibodies are present, which react with both the patient’s
own red blood cells and nearly all donor RBCs. Therefore,
serologically compatible blood is rarely available for
transfusion. The inability to provide crossmatch-compatible
blood has led to concerns that transfusion of these patients
could precipitate acute hemolysis with potentially severe
consequences (hemoglobinemia, hemoglobinuria, acute renal
failure, disseminated intravascular coagulation and death).
These concerns are based on a few published reports
of post-transfusion hemolysis in such cases. Because of
the limited amount of this data and anecdotal experience
suggesting a lack of severe adverse events after transfusion,
the indications for and advisability of transfusion in these
patients remain controversial issues.
Concern regarding the precipitation of acute hemolytic
transfusion reactions leading to morbidity and mortality after
transfusion of red blood cells in patients with autoimmune
hemolytic anemia stems from a few early reports of fatalities
in these patients. While a temporal relationship between
transfusion and hemoglobinemia/hemoglobinuria has been
seen in a handful of cases, a careful analysis of these reports
does not support a cause and effect relationship between
transfusion and death. The available safety and efficacy
data for transfusion in patients with autoimmune hemolytic
anemia, while somewhat limited, suggests that blood
transfusion is well-tolerated and efficacious.
Suggested reading
1. Kohan et al. Vox Sanguinis. 1994;67(2):195-8.
2. Mauro et al. Blood. 2000; 95(9):2786-92.
3. Narvios et al. Curr Issues Trans Med. 2001;9(3):1-5.
4. Shirey et al. Transfusion. 2002;42(11):1435-41.

Sugar Free Antibodies: A
New Treatment for Idiopathic
Thrombocytopenic Purpura and
Autoimmune Hemolytic Anemia
HS Sandhu, Ishwar Chouhan
Introduction
Autoimmune destruction of self-tissue is the consequential
result of a convergence of several factors, both genetic and
environmental, that effectively dislocates the immune system’s
ability to tolerate self-antigens but simultaneously retains its
focus on those perceived as foreign. Although many of these
autoimmune targeted self-antigens are known, immune system
triggers can be more elusive. Nevertheless, once autoimmunity
has been initiated, immune complexes (IC) play as a significant
driving force in maintaining chronic inflammation and
subsequently pathology in many of these conditions. ICs are
antibody/antigen assemblies, usually IgG antibody subtype,
sometimes found systemically but primarily at the site of
autoimmune self-antigen recognition.1 Sugar free antibodies
are deglycosylated IgG having a dominant suppressive effect
on inflammation and points to a unique class of therapeutic
immunoglobulins for the treatment of autoimmunity.
The enzyme EndoS from Streptococcus pyogenes is an
immunomodulatory molecule hydrolyzing the conserved
glycans in the effector part of immunoglobulin G (IgG).
EndoS is remarkably specific for IgG, and hydrolysis having
profound effects on IgG effector functions. EndoS pre-
treatment of IgG, or direct administration to animals with
experimental antibody-mediated autoimmune diseases,
inhibits development of disease or cures animals from
established disease. The properties of EndoS make it a unique
experimental tool and an attractive alternative to current
therapies of conditions involving pathogenic antibodies.2
Sugar Free Antibodies in Immune
thrombocytopenic purpura
Collin et al.3 adopt a novel experimental approach to this
challenge: enzymatic removal of carbohydrates from IgG Fc
in vivo. The enzyme chosen, EndoS, is isolated from a common
human pathogen Streptococcus pyogenes and specifically
hydrolyzes the Fc glycans between the two core GlcNAc
residues. This modification of Fc abolishes the binding of
IgG to Fc receptors and reduces complement activation.
3
In their study, Collin et al. first showed that EndoS, at low
concentration, efficiently hydrolyzes the glycan on IgG in the
complex environment of human blood in vitro. They then went
on to show that the hydrolysis could be achieved efficiently in
the circulation of a live animal by injecting rabbits with EndoS
and monitoring total IgG. Low concentrations of EndoS were
effective without perturbing the total IgG concentration or
disrupting other serum glycoproteins. Furthermore, the IgG
glycan hydrolysis effect was observed even in the face of a
host antienzyme response. To investigate whether the glycan
modification might have utility in modifying IgG activity,
they chose a mouse model of a human autoimmune disease,
immune thrombocytopenic purpura (ITP), in which platelets
are targeted by autoantibodies. In the model, polyclonal
antiplatelet IgG is injected into the mice to cause disease.
Collin et al. showed that pretreatment of the antiplatelet
IgG with EndoS or treatment of the mice with EndoS after
initiation of disease prevented lethal thrombocytopenia in
the majority of animals. Most impressively, even if disease
was allowed to proceed to symptoms, as might be the case
for humans presenting with ITP, EndoS treatment was able to
provide considerable benefit.3
How does EndoS Exercise a Protective
Function?
One plausible mechanism is that “activating” signals from
C1q, Fc_Rs, and MBL binding are all reduced or abolished,
whereas the interaction with the “inhibitory” Fc_RIIB
receptor is preserved. This mechanism may reflect the
natural immunosuppressive role of this enzyme. S. pyogenes
has evolved several mechanisms to evade and diminish the
adaptive immuneresponse, one of these appears to effectively
switch off the effector functions of IgG.
Sugar Free Antibodies in Autoimmune
hemolytic anemia
The presence and pathogenicity of autoantibodies directed
against red blood cells (RBCs) have been extensively
10  Section 1  Clinical Hemotherapy
investigated and implicated in a number of autoimmune
diseases, such as autoimmune hemolytic anemia (AIHA)
and systemic lupus erythematosus.4 The RBC surface
contains many epitopes recognizable by alloantibodies
and autoantibodies, including more than 300 blood group
antigens located on glycoproteins and glycolipids but also
nonpolymorphic/nonblood group protein structures.5 The
most common targets of pathogenic RBC autoantibodies
are epitopes on the Rh protein, encoded by 2 homologous
genes on chromosome 1, RHD and RHCE.6 More than half
of all autoantibody specificities in patients with AIHA of
the common so-called warm type include immunoglobulin
G (IgG) antibodies against these Rh epitopes, which are
consequently often used to model AIHA experimentally.
Human antibodies against RhD (anti-D) or rabbit
antibodies against human RBC (anti-RBC) can result in
accelerated phagocytosis and removal of RBC from the
circulation by monocytes/macrophages in the spleen and
liver.7,8 Fcγ receptor (FcγR)-mediated erythrophagocytosis,
in addition to other IgG effector functions, such as activated
complement, oxidative burst, and cytokine production by
FcγR expressing effectors cells, play an essential role in
shortened RBC survival.9-13 The ability of normal human
monocytes to bind and phagocyte IgG sensitized RBCs via
FcγR and mediate immune RBC destruction is highly related
to the glycosylation status of the Fc domain on the anti-RBC
antibodies.14-19
A study was undertaken to establish the effects of EndoS
on antibody-mediated destruction of RBCs with a future
therapeutic application in mind. Here it was shown, using
monocytes from blood and the THP-1 monocytic cell
line, that pretreatment of anti-RBC with EndoS abrogates
erythrophagocytosis and subsequent activation of monocytes.
The complement-dependent hemolytic effects of anti-RBC
added to whole human blood were greatly decreased by
pretreatment of this antibody with EndoS. Finally and most
importantly, EndoS showed inhibitory effects on a model of
AIHA in mice. These results suggest that anti-RBC antibodies
can be rendered less pathogenic by treatment with EndoS,
which may be regarded as a possible future inhibitor of
antibodymediated destruction of RBCs and AIHA.
Mechanism of Action
Treatment of Anti-D with EndoS does not
Affect the Binding of Anti-D to RBCs
EndoS inhibits binding of IgG to soluble and monocyte-
bound FcγR by hydrolyzing the N-linked glycan on the heavy
chain of IgG.20 In a study conducted by Allhorn M, the results
showed that the treatment of anti-D with EndoS did not affect
the antigen recognition by this antibody and the amount of
RBC-bound IgG was not influenced by EndoS treatment.
EndoS Inhibits Phagocytosis and Adherence of
Sensitized RBCs by Monocytes
The effect(s) of EndoS on phagocytosis of anti-D sensitized
RBCs by monocytes was analyzed using monocyte monolayer
assay. Monolayers of monocytes were cultured on slides,
and RBCs presensitized with anti-D were added to the
monolayers. The degree of phagocytosis was determined
using light microscopy and expressed as the number of
monocytes with adherent/phagocytosed RBCs by analyzing
100 monocytes. As expected, the phagocytosis of anti-D–
sensitized RBCs was significantly greater than nonsensitized
RBCs and was efficiently eliminated by pretreatment of anti-D
with EndoS (p < 0.001). This clearly shows that the treatment
of anti-D with EndoS before sensitization of RBCs abrogates
the phagocytosis-enhancing activity of anti-D and decreases
the accumulation of intracellular hemoglobin in monocytes.
EndoS Inhibits Oxygen Metabolite Production by
Monocytes Induced by Sensitized RBCs
In an attempt to further elucidate the effects of EndoS on
monocyte-driven cell cytotoxicity to sensitized RBCs, we
measured the release of oxygen metabolites from monocytes
using a chemiluminescence test. RBCs were opsonized with
immune plasma with high titers of anti-D from 3 persons.
Monocyte responses to sensitized RBCs were compared with
their response to unsensitized cells and expressed as a ratio
(opsonic index). RBCs incubated with immune plasma gave
positive results in chemiluminescence test with opsonic index
2.5. In contrast, monocyte responses to RBCs incubated with
immune plasma pretreated with EndoS were significantly
decreased (plasma 1, p < 0.07; plasma 2, p < 0.02; plasma 3,
p < 0.015) and in some cases even below the control plasma.
These results suggest that treatment of immune plasma with
EndoS abrogates in vitro metabolic oxidative responses by
monocytes.
EndoS Inhibits IL-8 Secretion from Monocytes
Induced by Sensitized RBCs
It has been reported that IL-8, besides being produced
by monocytes in response to lipopolysaccharide, tumor
necrosis factor-α, or IL-1, is implicated as an indicator
of RBC incompatibility and mediator of the pathologic
events in hemolysis in, for example, hemolytic transfusion
reactions.21-23 It has been shown that monocytes exposed to
sensitized RBC’s release IL-8, which was measured by ELISA
method, as expected a significant rise in levels of IL-8 were
observed. Noticeably, RBCs sensitized with anti-D pretreated
with EndoS showed significantly decreased secretion
of IL-8 in THP-1 cells (p < 0.001) and bloodmonocytes
(p < 0.003), which was comparable with the secretion of IL-8
 Chapter 3  Sugar Free Antibodies: A New Treatment for Idiopathic Thrombocytopenic Purpura...  11
by THP-1 cells and monocytes because of stimulation with
nonsensitized RBCs (p < 0.001 and p < 0.002, respectively.
These results demonstrate that EndoS does not have any
direct cytotoxic effects on monocytes.
EndoS Inhibits Hemolysis of Sensitized RBCs and
Prevents Binding of C1q to IgG-sensitized RBCs
Human anti-D antibodies of IgG isotype do not generally
activate the complement pathway, and RBCs coated with
these antibodies are preferentially cleared by mononuclear
phagocytic cells via FcγRs. It was clearly shown that pre-
treatment of antibody by EndoS is not a prerequisite for
inhibition of hemolysis and that a direct addition of EndoS
to blood was sufficient to exclude the hemolytic effect of anti-
RBC (p < 0.001). EndoS by itself did not have any effect on
hemolysis (p < 0.001). Furthermore, addition of increasing
concentrations of anti-RBC IgG to whole blood caused a
strong agglutination reaction as expected. Remarkably,
addition of EndoS-treated antibody abrogated this effect.
In conclusion, these data indicate that EndoS possesses
antihemolytic activities as demonstrated by pretreatment
of anti-RBC with enzyme or by a direct addition of EndoS to
anti-RBC-containing blood.
It has been reported that C1q, the recognition subunit
of the first component of the classic complement pathway,
binds to IgG-sensitized RBCs.24 Because the binding of C1q
to IgG is dependent on the glycosylation state of IgG, we
decided to explore the effect(s) of EndoS on deposition of C1q
onto IgG-sensitized RBCs and with this further elucidate the
mechanism for the antihemolytic effects of EndoS. There was
significantly decreased fluorescence intensity in the samples
containing RBCs sensitized with EndoS-treated anti-RBC
(p < 0.001) compared with RBCs treated with native antibody.
These results indicated an inability of C1q to bind to these
RBCs, providing further evidence for the complement-
inhibitory/antihemolytic properties of EndoS.
EndoS Inhibits AIHA In Vivo
The deposition of C1q and C3 on RBCs was analyzed by
flowcytometry 24 hours after injection of EndoS. This
revealed a significant decrease in C1q and C3 deposition on
RBCs in mice treated with EndoS compared with PBS-treated
mice (mean fluorescence intensity: C1q, 28.4 ± 6.5 vs 42.6
± 7.8, p < 0.02; C3, 13.1 ± 4.8 vs 29.6 ± 9.7, p < 0.01), suggesting
a reduced activation ofcomplement. The results showed that
there was a substantial reduction of iron deposits in livers
from mice treated with EndoS, compared with mice without
treatment of EndoS. In addition, the effect of EndoS on 34-3C
IgG1 and IgG2a class-switch variants was also determined. We
found protective effect of EndoS on IgG1 induced anemia but
not on IgG2a-induced anemia. When measuring the extent
of C1q and C3 deposits in mice injected with complement-
activating 34-3C IgG2a, no significant differences were
visible compared with mice without EndoS treatment. Data
indicate that EndoS partially inhibits development of anemia
mediated by anti-RBC reactive antibodies in an IgG subclass-
dependent manner in mice.
Sugar Free Antibodies in Other
Autoimmune Diseases
Nandakumar et al. used the mouse anticollagen type II (anti-
CII) mediated-arthritis model to show that deglycosylated
IgG, regardless of Fab antigen specificity, reduces inflammation
in a dose-dependent manner.1 This finding suggests that
deglycosylated IgG has a dominant suppressive effect on
inflammation and points to a unique class of therapeutic
immunoglobulins for the treatment of autoimmunity.1
Future of this Therapeutic Modality
The idea that antibody-mediated autoimmunity could be
therapeutically controlled by a single enzyme is certainly
an attractive one, but will Fc deglycosylation become a
therapeutic reality?
Several questions remain. EndoS is a bacterial protein and
is readily targeted for removal by antibodies, which would
normally limit the half-life of the protein in the blood. However,
Collin et al. report that, although antibodies to EndoS were
observed in their study, the enzyme nonetheless exhibited
reasonable pharmacokinetics. A possible explanation for this
observation is that the normal Fc-mediated mechanisms for
clearing foreign proteins are reduced, because the IgGs bound
to the enzyme are themselves deglycosylated by the enzyme.
Another potential obstacle might be that EndoS would cleave
glycans other than those on IgG with unknown consequences.
Encouragingly, however, EndoS appears unusually specific
for the glycans on IgG, probably a consequence of its intrinsic
affinity for Fc.
References
1. Nandakumar KS, et al. Dominant suppression of inflammation
by glycan-hydrolysed IgG. Pro Natl Acad Sci USA. 2013;
110:10252-7.
2. Allhorn M,  Collin M. Sugar-free  antibodies: the bacterial
solution to autoimmunity? Ann NY Acad Sci. 2009;1173:664-9.
3. Collin M, Shannon O, Björck L. Proc Natl Acad Sci USA.
2008;105:4265-70.
4. Kavai M, Szegedi G. Immune complex clearance by monocytes
and macrophages in systemic lupus erythematosus.
Autoimmun Rev. 2007;6(7):497-502. [PubMed: 17643939].
5. Daniels G. Naming blood groups and the genes that control
them. ISBT Science Series. 2009;4(1):118-20.
6. Suto Y, Ishikawa Y, Hyodo H, et al. Gene organization and
rearrangements at the human Rhesus blood group locus
12  Section 1  Clinical Hemotherapy
revealed by fiber-FISH analysis. Hum Genet. 2000;106(2):164-
71.[PubMed: 10746557].
7. Kumpel BM, Goodrick MJ, Pamphilon DH, et al. Human
Rh D monoclonal antibodies (BRAD-3 and BRAD-5)
cause accelerated clearance of Rh D+ red blood cells and
suppression of Rh D immunization in Rh D− volunteers.
Blood.1995;86(5):1701-9. [PubMed: 7655002].
8. Kávai M, Gyimesi E, Szucs G, et al. Binding and endocytosis of
erythrocytes sensitized with rabbit IgGvia Fc gamma receptors
of human monocytes. Immunology. 1991;74(4):657-60.
[PMCID: PMC1384775][PubMed: 1838354].
9. Schreiber AD, Frank MM. Role of antibody and complement in
the immune clearance and destruction of erythrocytes: In vivo
effects of IgG and IgMcomplement-fixing sites. J Clin Invest.
1972;51(3):575-82.[PMCID: PMC302163] [PubMed: 4536807].
10. Clynes R, Ravetch JV. Cytotoxic antibodies trigger inflammation
through Fc receptors. Immunity. 1995;3(1):21-6. [PubMed:
7621075].
11. Meyer D, Schiller C, Westermann J, et al. FcgammaRIII (CD16)-
deficient mice show IgG isotype-dependent protection to
experimental autoimmune haemolytic anemia. Blood.
1998;92(11):3997–4002. [PubMed: 9834201].
12. Flesch BK, Voge K, Henrichs T, et al. Fc gamma receptor-
mediated immune phagocytosis depends on the class of Fc
gamma R and on the immunoglobulin coated target cell. Vox
Sang. 2001;81(2):128-33.[PubMed: 11555474].
13. Davenport R. Cytokines and erythrocyte incompatibility. Curr
Opin Hematol. 1994;1(6):452-6. [PubMed: 9371322].
14. Hadley AG, Zupanska B, Kumpel BM, et al. The glycosylation of
red cell autoantibodies affects their functional activity in vitro.
Br J Haematol. 1995;91(3):587-94. [PubMed: 8555059].
15. Kumpel BM, Wang Y, Griffiths HL, et al. The biological
activity of human monoclonal IgG anti-D is reduced by
beta-galactosidase treatment. Hum Antibodies Hybridomas.
1995;6(3):82-8.[PubMed: 8597627].
16. Malaise MG, Hoyoux C, Franchimont P, et al. Effects of mannose
and mannose derivatives on the clearance of IgG antibody-
coated erythrocytes in the rat. Immunology. 1989;68(1):126-32.
[PMCID: PMC1385516] [PubMed: 2530153].
17. Malaise MG, Franchimont P, Mahieu PR. The ability of normal
human monocytes to phagocytose IgG-coated red blood cells
is related to the number of accessible galactosyl and mannosyl
residues in the Fc domain of the anti-red blood cell IgG
antibody molecules. J Immunol Methods. 1989;119(2):231-9.
[PubMed: 2723441].
18. Kaneko Y, Nimmerjahn F, Ravetch JV. Anti-inflammatory
activity of immunoglobulin G resulting from Fc sialylation.
Science. 2006;313(5787):670-3. [PubMed: 16888140].
19. Nimmerjahn F, Anthony RM, Ravetch JV. Agalactosylated
IgG antibodies depend on cellular Fc receptors for in vivo
activity. Proc Natl Acad Sci USA. 2007;104(20):8433-7. [PMCID:
PMC1895967][PubMed: 17485663].
20. Allhorn M, Olin AI, Nimmerjahn F, et al. Human IgG/Fc gamma
R interactions are modulated by streptococcal IgG glycan
hydrolysis. PLoS ONE. 2008;3:e1413. [PMCID: PMC2173940]
[PubMed: 18183294].
21. Davenport RD, Burdick MD, Strieter RM, et al. In vitro
production of interleukin-1 receptor antagonist in IgG-
mediated red cell incompatibility. Transfusion. 1994;34(4):297-
303. [PubMed: 8178326].
22. Davenport RD, Strieter RM, Standiford TJ, et al. Interleukin-8
production in red blood cell incompatibility. Blood.
1990;76(12):2439-42. [PubMed: 1702325].
23. Wikman A, Axdorph U, Gryfelt G, Gustafsson L, Björkholm
M, Lundahl J. Characterization of red cell autoantibodies
in consecutive DAT-positive patients with relation to in
vivo haemolysis. Ann Hematol. 2005;84(3):150-8. [PubMed:
15551098].
24. Basta M, Fries LF, Frank MM. High doses of intravenous
immunoglobulin do not affect the recognition phase of the
classical complement pathway. Blood. 1991;78(3):700-2.
[PubMed: 1859883].
Transfusion Practice in
Disseminated Intravascular
Coagulation
Hitish Narang
Disseminated intravascular coagulation (DIC) is character­i­
zed by systemic activation of blood coagulation, which results
in generation and deposition of fibrin, leading to microvascular
thrombi in various organs and contributing to multiple
organ dysfunction syndrome (MODS).1 Consumption and
subsequent exhaustion of coagulation proteins and platelets
(from ongoing activation of coagulation) may induce severe
bleeding, though microclot formation may occur in the
absence of severe clotting factor depletion and bleeding.2
Derangement of the fibrinolytic system further contributes
to intravascular clot formation, but in some cases, accelerated
fibrinolysis may cause severe bleeding. Hence, a patient with
disseminated intravascular coagulation (DIC) can present
with a simultaneously occurring thrombotic and bleeding
problem, which obviously complicates the proper treatment.
Definition of DIC by International Society on Thrombosis
and Hemostasis: “An acquired syndrome characterized
by the intravascular activation of coagulation with loss of
localization arising from different causes. It can originate
from and cause damage to the microvasculature, which if
sufficiently severe, can produce organ dysfunction”.3
Disseminated intravascular coagulation is not itself a
specific illness; rather, it is a complication or an effect of
the progression of other illnesses. It is always secondary to
an underlying disorder and is associated with a number of
clinical conditions, generally involving activation of systemic
inflammation such as:4 Sepsis, Trauma, Organ dysfunction,
malignancy, Severe transfusion reactions, obstetric complica­
tions, Vascular abnormalities, Hepatic failure and heat stroke.
Disseminated intravascular coagulation is estimated to be
present in approximately 1% of hospitalized patients.5
Pathophysiology
The hematologic derangements seen in DIC result from the
following four simultaneously occurring mechanisms:6
1. Tissue factor mediated thrombin generation.
2. Dysfunctional physiologic anti-coagulant mechanisms.
4
3. Impaired fibrin removal due to depression of fibrinolytic
system.
4. Inflammatory activation.
Diagnosis
The diagnosis of DIC is not made on a single laboratory
value, but rather the constellation of laboratory markers
and a consistent history of an illness known to cause DIC.
Laboratory markers consistent with DIC include:7–9
• Characteristic history
• Prolonged prothrombin time and activated partial
thromboplastin time
• Fibrinogen level
• Rapidly declining platelet count
• High levels of fibrin degradation products, including
D-Dimer
• Peripheral blood smear showing schistocytes.
Treatment
• Treatment of underlying condition
• Transfusion of platelets, fresh frozen plasma and packed
red cells
• Transfusion of cryoprecipitate when fibrinogen level is
low
• Treatment of Thrombosis with heparin, antithrombin and
antifibrinolytics is not usually used
• Recombinant factor VII.15
Administration of Blood Components and
Coagulation Factors
Platelet and coagulation factor replacement should not
be instituted on the basis of laboratory results alone; such
therapy is indicated only in patients with active bleeding and
in those requiring an invasive procedure or who are otherwise
at risk for bleeding complications.
14  Section 1  Clinical Hemotherapy
Platelets
Platelet transfusion may be considered in patients with DIC
and severe thrombocytopenia, in particular, in patients with
bleeding or in patients at risk for bleeding (e.g. in the early
postoperative phase or if an invasive procedure is planned).
The threshold for transfusing platelets varies. Platelet
replacement is usually required in non-bleeding patients
if platelet counts drop below 20 × 109/L. In some instances,
platelet transfusion is necessary at higher platelet counts,
particularly if indicated by clinical and laboratory findings.10
In actively bleeding patients, platelet levels from 20 × 109/L to
50 × 109/L are grounds for platelet transfusion (1 or 2 U/kg/
day).
Coagulation Factors
Cryoprecipitate and coagulation factor concentrates should
not routinely be used as replacement therapy in DIC, because
they lack several specific factors (e.g. factor V). Additionally,
worsening of the coagulopathy via the presence of small
amounts of activated factors is a theoretical risk. Specific
deficiencies in coagulation factors, such as fibrinogen, can
be corrected by administration of cryoprecipitate or purified
fibrinogen concentrate in conjunction with fresh frozen
plasma administration.
The consumption-induced deficiency of coagulation
factors can be partially rectified by administering large
quantities of fresh frozen plasma (FFP), particularly in
patients with an international normalized ratio (INR) higher
than 2.0, a 2-fold or greater prolongation of the aPTT, or a
fibrinogen level below 100 mg/dL.11 The suggested starting
dose is 15 mg/kg.12
Repeated measurement of global clotting tests (e.g. aPTT
and PT) might be useful for monitoring the coagulation
defect. In case of a (relative) vitamin K deficiency in the
face of consumption, administration of vitamin K may be
required.5,13,14
FFP: 10–20 mL/kg of body weight will increase factor levels
by 20–30%.
Frequency of transfusion depends on the half-life of the
deficient factor(s).
Prognosis
Prognosis varies depending on the underlying disorder and
the extent of intravascular thrombosis. The prognosis for DIC,
regardless of underlying cause, is often grim: between 10 and
50% patients will not survive. DIC with sepsis has a significantly
higher rate of death than DIC associated with trauma.16
References
1. Vincent JL, De Backer D. Does disseminated intravascular
coagulation lead to multiple organ failure? Crit Care Clin.
2005;21(3):469-77. [Medline].
2. Levi M, Ten Cate H. Disseminated intravascular coagulation.
N Engl J Med. 1999;341(8):586-92. [Medline].
3. [Guideline] Taylor FB Jr, Toh CH, Hoots WK, Wada H,
Levi M. Towards definition, clinical and laboratory criteria, and
a scoring system for disseminated intravascular coagulation.
Thromb Haemost. 2001;86(5):1327-30. [Medline].
4. Levi M, de Jonge E, van der Poll T. New treatment strategies
for disseminated intravascular coagulation based on current
understanding of the pathophysiology. Ann Med. 2004;36(1):
41-9. [Medline].
5. Matsuda T. Clinical aspects of DIC–disseminated intravascular
coagulation. Pol J Pharmacol. 1996;48(1):73-5. [Medline].
6. Franchini M, Lippi G, Manzato F. Recent acquisitions in the
pathophysiology, diagnosis and treatment of disseminated
intravascular coagulation. Thromb J. 2006;4:4. [Medline]. [Full
Text].
7. Levi M. “Disseminated Intravascular Coagulation”. Critical
Care Medicine. 2007;35(9):2191-5.
8. Hematology: Basic Principles and Practice, 6th edn. Elsevier
Saunders. 2012. ISBN 1437729282.
9. Levi M, Toh CH, et al. “Guidelines for the diagnosis and
management of disseminated intravascular coagulation”.
British Journal of Haematology. 2009;145(5):24-33.
10. Levi M, Opal SM. Coagulation abnormalities in critically ill
patients. Crit Care. 2006;10(4):222. [Medline]. [Full Text].
11. Wada H, Asakura H, Okamoto K, Iba T, Uchiyama T, Kawasugi
K, et al. Expert consensus for the treatment of disseminated
intravascular coagulation in Japan. Thromb Res. 2010;125(1):
6-11. [Medline].
12. Levi M, Toh CH, Thachil J, Watson HG. Guidelines for the
diagnosis and management of disseminated intravascular
coagulation. British Committee for Standards in Haematology.
Br J Haematol. 2009;145(1):24-33. [Medline].
13. Levi M. Disseminated intravascular coagulation: What's new?
Crit Care Clin. 2005;21(3):449-67. [Medline].
14. Levi M. The coagulant response in sepsis. Clin Chest Med.
2008;29(4):627-42, viii. [Medline].
15. Franchini M, Manzato F, Salvagno GL, et al. Potential role
of recombinant activated factor VII for the treatment of
severe bleeding associated with disseminated intravascular
coagulation: a systematic review. Blood Coagul Fibrinolysis.
2007;18(7): 589.
16. Becker, Joseph U, Charles RW. Disseminated intravascular
coagulation at eMedicine, 10 September 2009.
Transfusion Practice in Oncology
Devinder Singh Sandhu
ANEMIA
Background
Anemia is a common complication in patients with
malignancy. It can impair the patient's functional status,
diminish physiologic reserve, and cause fatigue that can be
disabling.
In addition to causing symptoms, the presence of
anemia has been linked to an adverse prognosis in several
malignancies. This has been attributed in part to a poorer
response to anticancer treatment, since ionizing radiation and
some forms of chemotherapy are dependent upon adequate
tissue oxygen levels for cytotoxicity. These observations have
been used to provide an additional rationale for aggressively
treating anemia in patients receiving cancer therapy.
Multiple Factors can Cause or Contribute to
Anemia in Patients with Malignancy
• As can occur with other chronic inflammatory conditions,
some cancer patients have an anemia that is a consequence
of the disease and unrelated to treatment. This type of
anemia is typically mild (hemoglobin level >10 g/dL),
and the symptoms may be difficult to distinguish from
those caused by the underlying malignancy. Occasionally,
Table 1: Comparison of risks and benefits of erythropoiesis-stimulating agents versus red blood cell transfusion for chemotherapy-related
anemia in patients with solid tumors
  Erythropoiesis stimulating agents
Risks Thrombotic events
Potentially decreased survival
Benefits Gradual improvement in hemoglobin/hematocrit
Gradual clinical improvement, avoidance of RBC transfusions in
some patients, net reduction in transfusion requirements
5
anemia may be more severe, impairing functional status,
diminishing physiologic reserve, and reducing quality of
life (QOL).
• Anemia is often exacerbated by myelosuppressive cancer
treatment, particularly in patients who are undergoing
intensive chemotherapy or combined modality treatment
with both chemotherapy and radiation therapy.1-5
• In addition to direct effects of the malignancy or its
treatment, other causes of anemia may coexist in these
patients (e.g. blood loss, hemolysis, deficiencies of iron,
folate, or vitamin B12). These causes should be actively
sought, and when present, treated appropriately.
Management of Anemia in Cancer Patients
Definitive therapy for cancer-related anemia is eradication
of the underlying malignancy. However, in many cases, this
is not possible and short-term red blood cell (RBC) support
may be needed.
• Transfusion: Red blood cell (RBC) transfusions are almost
universally successful in raising Hb levels. Transfusions
can often ameliorate the patient's symptoms rapidly and
improve health-related QOL. Exceptions include patients
unable to be transfused because of the presence of multiple
alloantibodies and those who refuse transfusions based on
religious beliefs. In such cases, erythropoietin stimulating
agents (ESAs) may be beneficial (Table 1).
Red blood cell transfusion
Transfusion reactions
Circulatory overload
Viral infection
Iron overload
Development of multiple alloantibodies
Rapid improvement in hemoglobin/hematocrit
Rapid clinical improvement
16  Section 1  Clinical Hemotherapy
• ESAs: Epoetin and darbepoetin: Clinical trials have
established that epoetin and darbepoetin are effective
in raising hemoglobin levels and decreasing transfusion
requirements in a substantial number of patients with
chemotherapy-induced anemia. Both ESAs appear to be
equivalent with regard to efficacy and safety.6,7
• The use of ESA should be discontinued after eight weeks if
the Hb has not increased by more than 1 to 2 g/dL or there
is no diminution in the need for RBC transfusion.6,7
• For all patients treated with ESAs, supplemental iron be
given to maintain a transferrin saturation ≥20% and a
serum ferritin ≥100 ng/mL.

Choice of Agent, Dose Titration, and THROMBOCYTOPENIA

Supplemental Iron
• The ESAs epoetin and darbepoetin appear to be similarly
effective. Darbepoetin may be more convenient, based
upon a lower frequency of administration.
• The approved starting dose of epoetin is 150 U/kg three
times weekly or 40,000 U weekly, subcutaneously; the
approved starting dose for darbepoetin is 2.25 micro­
gram/kg weekly or 500 micrograms every three weeks,
subcutaneously.
• The dose should be adjusted in each patient to maintain
the lowest hemoglobin level sufficient to avoid red cell
transfusions; dose modification guidelines are presented
in the table (Table 2).
Background
Thrombocytopenia is defined as a platelet count below the
150 × 109/L.8 Typically, platelet counts higher than 50 × 109/L
do not lead to clinical problems unless platelet dysfunction
coexists with the low count. Medical help is usually sought by
a patient with platelet counts less than 30 × 109/L, suffering
from spontaneous bruising and purpura or with continuous/
relatively long-lasting bleeding from injuries and wounds.
Clinically significant spontaneous bleeding does not usually
occur until the platelet count is less than 10 × 109/L.
Thrombocytopenia arises from three main causes:
Ineffective production of platelets by bone marrow,
accelerated destruction of platelets, or splenic sequestration.

Table 2:  Dosing guidelines for epoetin and darbepoetin in adults

  Three times weekly dosing Weekly dosing
Epoetin Alfa
Starting dose (adults) 150 units/kg subcutaneously (SC) three times weekly 40,000 units SC
Reduce dose by Hemoglobin reaches a level needed to avoid transfusion or increases >1 g/dL in any two-week period.
25% if
Withhold dose if Hemoglobin exceeds a level needed to avoid red blood cell (RBC) transfusion; restart at 25% below the previous
dose when the hemoglobin approaches a level where transfusions may be required.
Increase dose To 300 units/kg SC three times weekly, if response is not To 60,000 units SC weekly, if response is not
satisfactory (rise in hemoglobin<1 g/dL after four weeks satisfactory (rise in hemoglobin<1 g/dL after four
of therapy and remains below 10 g/dL) to achieve and weeks of therapy and remains below 10 g/dL) to
maintain the lowest hemoglobin level sufficient to avoid achieve and maintain the lowest hemoglobin level
the need for RBC transfusion. sufficient to avoid the need for RBC transfusion.
Discontinue if After completion of chemotherapy course or if after eight weeks of therapy there is no response as measured by
hemoglobin levels or if transfusions are still required.
  Weekly dosing Once every three weeks dosing
Darbepoetin Alfa
Starting dose (adults) 2.25 microgram/kg SC once weekly 500 micrograms SC every three weeks
Reduce dose by Hemoglobin exceeds a level needed to avoid transfusion, or increases >1 g/dL in any two-week period.
40% if
Withhold dose if The hemoglobin exceeds a level needed to avoid RBC transfusion; restart at 40% below the previous dose when
the hemoglobin approaches a level where transfusions may be required.
Increase dose To 4.5 microgram/kg, if response is not satisfactory N/A
(no reduction in transfusion requirements or rise in
hemoglobin by <1 g/dL after six weeks of therapy in the
absence of a RBC transfusion) to achieve and maintain the
lowest hemoglobin level sufficient to avoid the need for
RBC transfusion.
Discontinue if After completion of chemotherapy course or if after eight weeks of therapy there is no response as measured by
hemoglobin levels or if transfusions are still required.
 Chapter 5  Transfusion Practice in Oncology   17

Approach to the Patient with • Malignancies

• Possibility of pregnancy
Thrombocytopenia9 (Flow chart 1)
• Recent travels (e.g. exposure to malaria, rickettsiosis,
The most important aspects that should be investigated dengue fever)
include the following: • Recent transfusions
• Presence of a family history of thrombocytopenia • Ingestion of alcohol
• Disease history, paying special attention to recent viral • Dietary habits
and bacterial infections; vaccinations • Risk factors for HIV and viral hepatitis.

Flow chart 1  Thrombocytopenia: Diagnostic algorithm starting with complete blood count

Abbreviations:  CAMT, congenital amegakaryocytic thrombocytopenia: CBC, complete blood count; DITP, drug-induced immune
thrombocytopenia; FTLS, familial thrombocytopenis-leukemia syndrome; HIT, heparin-induced thrombocytopenia; HIV, human immunodefici­
ency virus; HCV, hepatitis C virus; ITP, immune thrombocytopenia; TAR, thrombocytopenia-absent radius syndrome; VCF, velocardiofacial
syndrome, WAS, Wiskott-Aldrich syndrome.
18  Section 1  Clinical Hemotherapy
There are two types of drug-induced thrombocytopenia:10
1. By direct myelosuppressive effect (e.g. chemotherapy-
induced thrombocytopenia).
2. By immune-mediated destruction of platelets due to
an idiosyncratic drug reaction (drug induced immune
thrombocytopenia [DITP]).
Complete Blood Count (CBC)
Isolated thrombocytopenia is typically associated with
immune-mediated disorders11 and inherited disorders
(e.g. Bernard-Soulier, TAR syndrome), but is uncommon in
malignant processes involving bone marrow.
Thrombocytopenia associated with anemia and leuco­
penia (pancytopenia) can be caused by bone marrow sup­
pression by various medications (commonly chemotherapy,
rarely antihypertensive medications and antibiotics e.g.
chloramphenicol); viral infections (HIV); bacterial infections
(e.g. leishmaniasis); severe folate and B12 deficiency;
paroxysmal nocturnal hemoglobinuria; systemic lupus
erythematosus (SLE); inherited disorders (e.g. dyskeratosis
congenita, Fanconi anemia); malignancy (metastatic disease,
leukemia, lymphoma with bone marrow involvement, multiple
myeloma, rarely solid tumors) or bone marrow failure (e.g.
aplastic anemia, myelodysplastic syndrome).
Additional Investigations
• Elevated blood lactate dehydrogenase (LDH) and indirect
bilirubin, combined with low haptoglobin level and a
positive direct antiglobulin test (direct Coombs test).
• Elevated LDH combined with renal function impairment
may indicate TTP or hemolytic uremic syndrome (HUS).
• Blood coagulation tests disturbance: Prolonged prothrom-
bin time (PT), low fibrinogen and elevated D-dimers are
typical for DIC.
• Elevated liver enzymes with or without elevated bilirubin,
LDH and alkaline phosphatase should lead to the investi­
gation of hepatic disease (viral hepatitis, drug induced
or toxic hepatitis), cirrhosis, and viral infection such as
cytomegalovirus (usually only liver enzymes and LDH are
elevated).
• Serological tests for viruses, blood cultures, anti-platelet
antibodies, bone marrow biopsy and many other
diagnostic tests can be performed at the discretion of the
physician according to the presenting signs and course of
the disease.
Treatment of Thrombocytopenia
In a case of severe bleeding, if the etiology of thrombocytopenia
is unknown, but not thought to be immunologic, platelet
transfusion can be used to provide an immediate platelet
increase. In contrast, if the underlying cause is immune,
the effect from platelet transfusion may be minimal and
at best very short-lived, and it should be reserved only
for life-threatening bleeding (ideally transfused following
intravenous immunoglobulin to “protect” the platelets).
If the cause of thrombocytopenia is unknown and there
are no contraindications, such as infections, corticosteroids
may be used to increase the platelet count.
NEUTROPENIA
Background
Neutropenia, usually defined as an absolute neutrophil
count (ANC) below 1.5 × 109/L (1500/mm3), encompasses
a wide range of diagnoses, from normal variants to life-
threatening, acquired and congenital disorders. The
functional consequences depend largely, but not exclusively,
on the severity of neutropenia: ANC of 1.0–1.5 × 109/L does
not impair host defense, but may warrant investigation of the
underlying cause; ANC of 0.5–1.0 × 109/L may slightly increase
the risk of infections, but only if other arms of the immune
system are impaired; ANC of 0.2–0.5 × 109/L is associated with
an increased risk of infections in most patients. ANC of 0.2 ×
109/L or less carries a risk of severe, life-threatening infections
with susceptibility to opportunistic organisms.12,13
Classification of Neutropenia
Neutropenia can be described as transient (or “acute”)
(Table 3) or chronic (or “persistent”) (Table 4); extrinsic
or intrinsic; by descriptive names (e.g. neonatal isoimmune
neutropenia of infancy, cyclic neutropenia, severe congenital
neutropenia) and as syndromes (e.g. Kostmann, Shwachman-
Diamond, and Barth syndromes).14,15
Table 3: Causes of transient (acute) neutropenia (other than
chemotherapeutic agents)
  Infection
Viral Cytomegalovirus, Epstein-Barr virus, HIV,
influenza, parvovirus
Bacterial Brucella, paratyphoid, tuberculosis,
tularemia, typhoid; Anaplasma
phagocytophilum and other rickettsia
Protozoan Plasmodium vivax, Plasmodium falciparum
Drugs
Anticonvulsant Carbamazepine, valproate
Antimicrobial Sulfonamides, penicillins, trimethoprim/
sulfamethoxazole
Antipsychotic Clozapine, olanzapine, phenothiazines
Antirheumatic Gold, levamisole, penicillamine
Antithyroid Methimazole, propylthiouracil
Other Aminopyrine, deferiprone, rituximab,
levamisole-adulterated cocaine
Immune
Neonatal isoimmune
Autoimmune

Table 4: Causes of persistent (chronic) neutropenia
Extrinsic Vitamin B12, folate, copper, protein-calorie
Nutritional Autoimmune
Immune Congenital immunological disorder
Systemic autoimmune disorder
Intrinsic
Myelodysplasia
Acquired bone Aplastic anemia
marrow failure
Congenital bone
marrow failure
Isolated Severe congenital neutropenia cyclic
neutropenia neutropenia
Neutropenic Disorders of granule sorting: Chediak-Higashi
syndromes syndrome, Cohen syndrome, Griscelli syndrome
type 2 Hermansky-pudlak syndrome type 2 and
p14 deficiency
Disorders of metabolism: Glycogen storage
disease type 1b, Barth syndrome, Pearson
syndrome
Disorders of immune function: Hyper-IgM
syndrome, WHIM syndrome, cartilage-hair
hypoplasia Schimke immuno-osseous dysplasia
Disorders of molecular homeostasis: Dyskeratosis
congenita, Fanconi anemia, Shwachman-
Diamond syndrome
Idiopathic
Management of Isolated Neutropenia
Acute infections: Patients with ANC of 0.2 × 109/L or
less almost invariably require hospital admission for IV
antibiotics, with the choice of drugs depending upon local
community and/or hospital flora and antibiotic sensitivities.
Importantly, antibiotic therapy should include anaerobic
coverage when fever is accompanied by abdominal pain,
as may recur frequently in cyclic neutropenia. Patients with
an ANC 0.2 ×109/L usually require admission, but culturing
and out-patient antibiotics may be used for some with more
benign forms of neutropenia, such as chronic autoimmune
neutropenia of infancy, with relatively good delivery of
neutrophils to tissues sites of infection.
Chronic Neutropenia
Individuals with the diagnosis of chronic neutropenia
who maintain ANC>1.0 × 109/L on G-CSF therapy may
be evaluated and treated as would any non-neutropenic
patients. Those in the intermediate range of ANC 0.5–1.0 ×
109/L need evaluation on a case-by-case basis.
Myeloid Growth Factor Therapy
Hematopoiesis is regulated by a family of hematopoietic
growth factors; the myeloid specific cytokine granulocyte
Chapter 5  Transfusion Practice in Oncology   19
colony-stimulating factor (G-CSF) is the principal regulator
of the blood neutrophil count. Recombinant human G-CSF
(filgrastim) is now widely used to stimulate neutrophil
production in patients with chemotherapy-induced
neutropenia. It is also very useful for management of severe
chronic neutropenia.16
References
1. Cash JM, Sears DA. The anemia of chronic disease: spectrum
of associated diseases in a series of unselected hospitalized
patients. Am J Med. 1989;87:638.
2. Schilling RF. Anemia of chronic disease: a misnomer. Ann
Intern Med. 1991;115:572.
3. Lind M, Vernon C, Cruickshank D, et al. The level of
haemoglobin in anaemic cancer patients correlates positively
with quality of life. Br J Cancer. 2002;86:1243.
4. Samol J, Littlewood TJ. The efficacy of rHuEPO in cancer-
related anaemia. Br J Haematol. 2003;121:3.
5. Straus DJ, Testa MA, Sarokhan BJ, et al. Quality-of-life and
health benefits of early treatment of mild anemia: a randomized
trial of epoetinalfa in patients receiving chemotherapy for
hematologic malignancies. Cancer. 2006;107:1909.
6. Rizzo JD, Brouwers M, Hurley P, et al. American Society of
Hematology/American Society of Clinical Oncology clinical
practice guideline update on the use of epoetin and darbepoetin
in adult patients with cancer. Blood. 2010;116:4045.
7. http://www.uptodate.com.
8. Lab Tests onlineUK: Platelet count aka thrombocyte count.
9. Provan D, Stasi R, Newland AC, Blanchette VS, Bolton-
Maggs P, Bussel JB, et al. International consensus report
on the investigation and management of primary immune
thrombocytopenia. Blood. 2010;115:168-86.
10. Cines DB, Liebman H, Stasi R. Pathobiology of secondary
immune thrombocytopenia. SeminHematol. 2009;46:S2-14.
11. Medscape reference library. [http://www.medscape.com/].
12. Hsieh MM, Everhart JE, Byrd-Holt DD, et al. Prevalence of
neutropenia in the US population: age, sex, smoking status,
and ethnic differences. Ann Intern Med. 2007;146:486-92.
[PubMed: 17404350].
13. Dale DC. Neutropenia and neutrophilia. In: Lichtman MA,
Kipps TJ, Kaushansky K, Beutler B, Seligsohn U, Prchal JT,
(Eds). Williams Hematology. New York: Mcgraw-Hill; 2010.
pp.939-50.
14. Husain EH, Mullah-Ali A, Al-Sharidah S, et al. Infectious etio-
logies of transientneutropenia in previously healthy children.
Pediatr Infect Dis J. 2012;31:575-7. [PubMed:22414904].
15. Dale DC, Bolyard AA, Schwinzer BG, et al. The Severe Chronic
Neutropenia InternationalRegistry: 10-Year follow-up report.
Support Cancer Ther. 2006;3:220-31. [PubMed: 18632498]
Newburger and Dale Page 10 SeminHematol. Author
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16. Dale DC, Bonilla MA, Davis MW, et al. A randomized
controlled phase III trial of recombinanthuman granulocyte
colony-stimulating factor (filgrastim) for treatment of severe
chronicneutropenia. Blood. 1993;81:2496–502. [PubMed:
8490166].
Transfusion Practice in Coronary
Interventions
Sudhir Varma
Introduction
Coronary care has taken a quantum leap after the advent of
angioplasty. Current pharmacoinvasive approach during
angioplasty permits use of a number of antithrombotic drugs.
Major bleeding after percutaneous coronary intervention
(PCI) is associated with increased mortality.1–6 The nature
of this relationship and its implications still remain unclear.
Is bleeding a surrogate for high-risk patients, because those
patients are at increased risk for bleeding complications too?
Bleeding may be an important, but until now overlooked,
cause of excess mortality following PCI. There is now robust
data that implicates direct and indirect effects of bleeding on
subsequent adverse events.7,8 Growing suspicion also has
focused on potentially harmful effects of red blood cell (RBC)
transfusion.1,4,5,8
The scope of the triad of bleeding, blood transfusion and
morality is beyond academic debates and no longer limited to
discussions. Major bleeding is dependent not only on patient
characteristics5 but also to a large extent on choice of vascular
access strategy (radial vs. femoral),9,10 and to a lesser extent on
the choice of an antithrombotic regimen.11 Earlier neglected,
inclusion of bleeding as a part of the primary end point in
some recent PCI trials has highlighted it as a major issue,
drawing equivalence between this complication and other
major adverse events (such as periprocedural myocardial
infarction) in the evaluation of novel therapeutic strategies in
coronary interventions.
Data from more than 90,000 patients are now
available, derived from a combination of unselected1,5–8
and randomized trial populations.2–4 Although bleeding
definitions and patient characteristics varied, a consistent
finding of increased mortality among patients who experience
major bleeding has emerged. Blood transfusion risk is there
beyond usual concerns. Emerging evidence suggests that
transfusion in PCI settings may be associated with risks over
and above usual concerns regarding microbial transmission
and acute antigen-antibody reactions.12,13 A number of
studies have examined the impact of RBC transfusion on
long-term outcomes after PCI and found a deleterious effect.
6
Among high-risk CAD patients with anemia8 or severe
bleeding,4 blood transfusion was required in >20% and was
associated with increased in-hospital mortality (adjusted
odds ratio: 2.02, p < 0.001)8 and increased 1-year mortality
(relative risk: 2.03, p = 0.028).4
Collectively, these studies demonstrate a robust
association among major bleeding, blood transfusion, and
increased mortality in patients undergoing PCI.4,8 They
suggest decreased survival among patients with acute
coronary syndromes who experience major bleeding6 or
require blood transfusion.6,11
It is important to emphasize, however, that none of these
studies can establish a causal connection between bleeding
events (or blood transfusion) and increased risk of death
after PCI.2,6 Although the relationship between bleeding and
mortality is described in several studies as an independent
one, many of the correlates of bleeding (such as advanced age
and renal failure) are themselves associated with mortality. As
a result, resolving the question of association versus causality
has been a tremendous challenge, but emerging mechanistic
data have begun to warm up the debate.
Why Bleeding and Excess Morality
POST Percutaneous coronary
intervention?
The biological impact of bleeding after PCI is likely to be
multifaceted. The location (intracranial) or torrential
nature of the hemorrhage (gastrointestinal, retroperitoneal)
may result in death, regardless of any previous cardiac
procedure. However, other consequences of bleeding
may have specific detrimental effects in patients who have
recently undergone coronary intervention and may offer an
explanation for the association between femoral bleeding
complications and mortality.14-16
Interactions between activated platelets and the
clotting cascade produce a rapid hemostatic response at the
site of vascular injury. Systemic amplification of this localized
response is prevented by a number of mechanisms, including
 Chapter 6  Transfusion Practice in Coronary Interventions  21
control of the activated procoagulants and platelets by
antithrombotic pathways.12 These antithrombotic pathways
are predominantly active within vascular endothelial cells,
but little is known about the function of these pathways
in patients with cardiovascular disease. Deficiency of
such antithrombotic protective mechanisms (in tandem
with systemic endothelial dysfunction) could lead to a
hypercoagulable state induced by bleeding, which would
pose obvious risks to the patient who had recently undergone
PCI.
Furthermore, experimental data suggest that increased
synthesis and release of erythropoietin in response to anemia
caused by bleeding might sustain a systemic prothrombotic
state beyond the acute phase, by causing platelet activation
and inducing plasminogen activator inhibitor-1 (a
procoagulant cytokine).3 Treatment with erythropoietin
has been associated with increased risk of thrombosis in
critical care patients.23 Finally, aspirin, clopidogrel, or other
antithrombotic medication may have to be discontinued
after a major bleeding event, thus increasing the risk for stent
thrombosis, a complication that frequently is fatal.14
The significance of these issues in everyday clinical practice
is difficult to quantify, but recent data have confirmed an
increased risk of acute ischemic events after major bleeding
suggesting that the hypothetical concerns may be justified.
Can Blood Transfusion in
Percutaneous coronary intervention
Patients be Harmful ?
Impairment oxygen delivery to the tissues is an important
issue. Hemoglobin level elevation can occur post blood
transfusion but paradoxically whereas transfusion increases
oxygen delivery, but measures of tissue oxygenation either
decrease or do not change.16 The reason for this paradox
(greater oxygen delivery but no increase in tissue use) is
unclear.18 Stored RBCs are low in 2, 3-diphosphoglyceric
acid (2, 3-DPG); therefore, the hemoglobin has high oxygen
affinity (i.e. will tend not to release oxygen to the tissues).
Traditionally, this finding been regarded as the pre-eminent
mechanism underlying reduced tissue extraction of oxygen
from the circulation after blood transfusion.17 However, 2,
3-DPG levels partially recover within several hours after
transfusion, and the results of experimental studies now
indicate that the physiologic impact of 2, 3-DPG depletion
may have been overstated.18 Recent data suggest that other
structural and biochemical changes that occur in RBCs
during storage might have a greater impact on their function
in vivo.15,28,29
Recent data indicate that this biochemical function is
significantly disrupted by storage of RBCs, with a rapid
decrease in S-nitrosothiol hemoglobin (SNO-hemoglobin)
concentrations to 30% of initial levels observed within
1 day of storage (>20% within 1 week).19 It is important to note
that it takes 48 hour from the time of donation to complete
the required testing and preparation of a unit of RBCs for
transfusion. Hypoxic vasodilation by banked RBCs correlated
strongly with the amount of SNO-hemoglobin, underscoring
the physiologic significance of the findings.20
Prothrombotic effects are noted with blood trans­fusions.
The transfusion of blood products has been associated with
an acute platelet release of CD40 ligand.21 Transfusion also is
associated with increased exposure to another procoagulant
protein, plasminogen activator inhibitor (PAI)-1. There is a
3- to 6-fold increase in PAI-1 content of packed RBCs stored
for greater than 35 days,22 and serum levels of PAI-1 increase
after allogeneic blood transfusion.23 Adenosine diphosphate,
a well-recognized mediator of platelet activation, might
also participate in transfusion related vascular thrombosis.
Adenosine diphosphate is released from activated platelets
and damaged endothelial cells and is also released by
erythrocytes in stored blood.24 Finally, free NO acts not only
as a potent vasodilator but also as an inhibitor of platelet
activation.21 Deficiencies of NO transport and release by
stored RBCs may therefore not only impact adversely on
regional blood flow in zones of hypoxia but also on risk for
thrombosis at sites of endothelial dysfunction.
Other adverse effects of blood transfusion. Trans­
fusion related immunomodulation (TRIM) is an immuno­
suppressive, or in some cases proinflammatory, effect
of blood transfusion that may contribute to adverse
outcomes among recipients of blood.21 The specific effects
of this phenomenon on plaque biology, thrombosis, and
microvascular function are as yet undefined, but given the
inflammatory nature of atherosclerosis, the potential for such
interplay is of obvious concern. Finally, severe hemolytic
reactions and the consequences of large-volume transfusion
(such as coagulopathy and electrolyte disturbance) also may
increase mortality associated with blood transfusion.
Duration of storage of transfused blood. Many of the
aforementioned concerns ultimately appear to hinge upon
the duration that blood is kept in storage before use. The
Food and Drug Administration USA allows packed red cells
to be stored for up to 42 days, allowing blood centers the
flexibility to manage this scarce resource but also influencing
the quality of blood that is transfused. Whether clinical
outcomes are adversely affected by longer storage, however,
is still uncertain. No data are available currently regarding
the impact of storage duration on outcomes for patients who
require transfusion after PCI. There are, however, reasonable
grounds for concern that administration of blood that has
been stored for longer than 14 days also may be associated
with worse outcomes for PCI patients, given the similarities
in clinical characteristics with cardiac surgery patients.
Implications for clinical practice. The concerns
outlined herein should not lead clinicians to withhold blood
transfusion when it is clearly indicated (i.e. severe anemia
causing symptoms or other evidence of ischemia, especially
22  Section 1  Clinical Hemotherapy
for patients who exhibit signs of oxygen-supply dependency).
Efforts to reduce the impact of bleeding complications on the
PCI population should rather focus on: (1) the identification
of measures to decrease the incidence of bleeding
complications without increasing risk for ischemic events;
and (2) the development of strategies for more targeted and
safer use of blood transfusion.
A prognostic risk score for major bleeding in patients
undergoing PCI via the femoral approach has been
published, allowing the identification of high-risk patients
before the procedure.25 Seven variables were proposed based
on an analysis of bleeding events from the REPLACE-2 trial:
age ≥ 55 years, female sex, estimated glomerular filtration
rate ≤ 60 mL/min/1.73 m2, pre-existing anemia, use of low-
molecular Weight heparin within 48 hour before PCI, use of
glycoprotein IIb/IIIa inhibitors, and the use of the intra-aortic
balloon pump2 Weighting for each was assigned by the use of
different integer scores, with a total score of >10 associated
with a major bleeding risk of >5%. Although this risk scoring
system remains to be validated in real-world cohorts, such a
tool may help inform decisions regarding initial management
strategy (for patients with borderline indications for PCI) and
enable the interventionalist to tailor the strategic approach
according to bleeding risk for those who do proceed with
intervention.
The single most effective way for the operator to reduce
major bleeding is to use radial rather than femoral access.10
A systematic review of randomized trials revealed an odds
ratio of 0.20 (95% confidence interval: 0.09 to 0.42; p < 0.0001)
for access-site complications after radial rather than femoral
PCI.14 Furthermore, in a retrospective study of almost 39,000
procedures, transradial PCI was associated with one-half
the number of bleeding complications, markedly reduced
transfusion requirements, and lower adjusted 30-day and
1-year mortality (p > 0.001).9 Selection bias could certainly
account for the finding of decreased mortality, as it seems
likely that the most complex cases requiring large devices
and hemodynamic support would have been performed
from the femoral route. However, it is possible that decreased
bleeding complications (and transfusion requirements) also
could have contributed, at least in part, to the finding of lower
mortality among patients treated via the radial artery.
Refinements in the use of blood transfusion. In addition
to reducing the incidence of major bleeding complications,
refinements in the use of blood transfusion may lead to
better outcomes for patients undergoing PCI. Appropriate
indications for transfusion after PCI have not yet been
defined, specifically with regard to the hemoglobin level
above which the risks of transfusion might outweigh any
benefit. Among patients with critical illness, a randomized
trial has demonstrated that those treated with the use of a
restrictive transfusion policy had better outcomes (when
compared with a more liberal transfusion policy),26 although
older patients with cardiac disease these findings might not
be applicable.
The situation is likely to be even more complex among
patients undergoing PCI, because factors such as clinical
presentation and completeness of revascularization will
probably alter the risk/benefit ratio for transfusion.
Baseline anemia is associated with increased need
for blood transfusion and greater mortality after PCI.27
Aggressive strategies to increase hemoglobin levels with
the use of iron repletion, erythropoietin, or other disease-
specific measures would probably reduce the need for
transfusion by providing a greater window of tolerance for
bleeding. It is reasonable to assume that such an approach
could also improve tolerance of myocardial ischemia during
long, complex cases. Although the value of postponing
elective PCI to treat anemia has not yet been tested, given
the frequency of this finding in routine clinical practice
such a study would clearly be of tremendous interest. There
are ongoing efforts to improve the quality of transfused
blood through refined harvest and storage techniques. For
example, decreased deformability of stored RBCs may be
ameliorated by correction of intracellular pH and restoration
of adenosine triphosphate levels.28 The immune response
to TRIM is attenuated by leukodepletion of blood products
before transfusion, suggesting that either a component or
byproduct of donor leukocytes elicits the recipient response.29
The age of RBCs at the time of collection might also affect the
impact of storage time on RBCs. In normal circumstances,
erythrocytes have a lifespan of approximately 120 days.
It would be expected therefore that a unit of blood would
contain a proportion of cells that are approaching the end
of their lifespan and may be more prone to storage induced
changes than younger cells. With a growing understanding
of storage-related hazards and the development of assays to
measure these, it appears likely that quality criteria for RBCs
will expand in the future to include functional biochemical
properties in addition to current regulations based on hemo­
lysis and hemoglobin mass. On the basis of current data the
use of arbitrary cutoffs (such as a hemoglobin of >8 g/dL)
to trigger transfusion after PCI should be avoided in most
circumstances. Risks and potential benefits of transfusion
should be weighed on clinical grounds.25,29 Among patients
with low hemoglobin who exhibit evidence of ischemia
despite successful revascularization, there is clear potential
for blood transfusion to have a net beneficial effect.27 When
there is clearly no evidence of ongoing ischemia, and therefore
minimal potential gain, adverse effects of transfusion may be
more likely to predominate (an exception to this may be the
asymptomatic patient who is considered at very high-risk
for rebleeding from a noncompressible site, where a greater
reserve of red cell mass may be desirable).
Conclusion
There is a strong association of major bleeding, blood
transfusion, and the risk of death after PCI. It remains to be
proven, however, that strategies to reduce bleeding and/or
 Chapter 6  Transfusion Practice in Coronary Interventions  23
blood transfusion will consistently lower all cause mortality.
The development of risk algorithm for bleeding following PCI
can be useful strategy for preventing such events. Despite
frequent use of blood transfusion in PCI patients, vexing
issues like thresholds for blood transfusion and blood storage
remain and need to be addressed to.
References
1. Kinnaird TD, Stabile E, Mintz GS, et al. Incidence, predictors,
and prognostic implications of bleeding and blood transfusion
following percutaneous coronary interventions. Am J Cardiol.
2003;92:930-5.
2. Feit F, Voeltz MD, Attubato MJ, et al. Predictors and impact
of major hemorrhage on mortality following percutaneous
coronary intervention from the REPLACE-2 trial. Am J Cardiol
2007;100:1364-9.
3. Ndrepepa G, Berger PB, Mehilli J, et al. Periprocedural bleeding
and 1-year outcome after percutaneous coronary interventions:
appropriateness of including bleeding as a component of a
quadruple end point. J Am Coll Cardiol. 2008;51:690-7.
4. Kim P, Dixon S, Eisenbrey AB, O’Malley B, Boura J, O’Neill
W. Impact of acute blood loss anemia and red blood cell
transfusion on mortality after percutaneous coronary
intervention. Clin Cardiol. 2007;30:II35-43.
5. Doyle BJ, Ting HH, Bell MR, et al. Major femoral bleeding
complications after percutaneous coronary intervention:
incidence, predictors, and impact on long-term survival
among 17,901 patients treated at the Mayo Clinic from 1994 to
2005. J Am Coll Cardiol Intv. 2008;1:202-9.
6. Eikelboom JW, Mehta SR, Anand SS, Xie C, Fox KA, Yusuf S.
Adverse impact of bleeding on prognosis in patients with acute
coronary syndromes. Circulation. 2006;114:774-82.
7. Spencer FA, Moscucci M, Granger CB, et al. Does comorbidity
account for the excess mortality in patients with major bleeding
in acute myocardial infarction? Circulation 2007;116:2793-
801.
8. Jani SM, Smith DE, Share D, et al. Blood transfusion and
in-hospital outcomes in anemic patients with myocardial
infarction undergoing percutaneous coronary intervention.
Clin Cardiol. 2007;30:II49-56.
9. Chase AJ, Fretz EB, Warburton WP, et al. The association
of arterial access site at angioplasty with transfusion and
mortality: the MORTAL Study: (Mortality benefit of Reduced
Transfusion After PCI via the Arm or Leg). Heart. 2008;94:1019-
25.
10. Agostoni P, Biondi-Zoccai GG, de Benedictis ML, et al. Radial
versus femoral approach for percutaneous coronary diagnostic
and interventional procedures; systematic overview and meta-
analysis of randomized trials. J Am Coll Cardiol. 2004;44:349-
56.
11. Lincoff AM, Kleiman NS, Kereiakes DJ, et al. Long-term efficacy
of bivalirudin and provisional glycoprotein IIb/IIIa blockade
vs heparin and planned glycoprotein IIb/IIIa blockade
during percutaneous coronary revascularization: REPLACE-2
randomized trial. JAMA 2004;292:696-703.
12. Lane DA, Philippou H, Huntington JA. Directing thrombin.
Blood. 2005;106:2605-12.
13. Taylor JE, Henderson IS, Stewart WK, et al. Erythropoietin and
spontaneous platelet aggregation in haemodialysis patients.
Lancet. 1991;338:1361-2.
14. McFadden EP, Stabile E, Regar E, et al. Late thrombosis in drug-
eluting coronary stents after discontinuation of antiplatelet
therapy. Lancet. 2004;364:1519-21.
15. Greenburg AG. A physiologic basis for red blood cell transfusion
decisions. Am J Surg. 1995;170:44S-8S.
16. Rao SV, Jollis JG, Harrington RA, et al. Relationship of blood
transfusion and clinical outcomes in patients with acute
coronary syndromes. JAMA 2004;292:1555-62.
17. Welch HG, Meehan KR, Goodnough LT. Prudent strategies for
elective red blood cell transfusion. Ann Intern Med. 1992;116:
393-402.
18. 2,3-diphosphoglycerate following transfusion of DPG-depleted
AS-1, AS-3 and CPDA-1 red cells. Br J Haematol. 1989;71:131-6.
19. Almac E, Ince C. The impact of storage on red cell function
in blood transfusion. Best Pract Res Clin Anaesthesiol.
2007;21:195-208.
20. Reynolds JD, Ahearn GS, Angelo M, Zhang J, Cobb F, Stamler
JS. S-nitrosohemoglobin deficiency: a mechanism for loss of
physiological activity in banked blood. Proc Natl Acad Sci USA.
2007;104:17058-62.
21. Twomley KM, Rao SV, Becker RC. Proinflammatory,
immunomodulating, and prothrombotic properties of anemia
and red blood cell transfusions. J Thromb Thrombolysis.
2006;21:167-74.
22. Nielsen HJ, Reimert C, Pedersen AN, et al. Leucocyte-derived
bioactive substances in fresh frozen plasma. Br J Anaesth.
1997;78:548-52.
23. Hedstrom M, Flordal PA, Ahl T, et al. Autologous blood
transfusion in hip replacement. No effect on blood loss but less
increase of plasminogen activator inhibitor in a randomized
series of 80 patients. Acta Orthop Scand. 1996;67:317-20.
24. Koch CG, Li L, Sessler DI, et al. Duration of red-cell storage
and complications after cardiac surgery. N Engl J Med
2008;358:1229-39.
25. Nikolsky E, Mehran R, Dangas G, et al. Development and
validation of a prognostic risk score for major bleeding in
patients undergoing percutaneous coronary intervention via
the femoral approach. Eur Heart J. 2007;28:1936-45.
26. Hebert PC, Wells G, Blajchman MA, et al. for the Transfusion
Requirements in Critical Care Investigators, Canadian Critical
Care Trials Group. A multicenter, randomized, controlled
clinical trial of transfusion requirements in critical care. N Engl
J Med. 1999;340:409-17.
27. McKechnie RS, Smith D, Montoye C, et al. Prognostic implication
of anemia on in-hospital outcomes after percutaneous
coronary intervention. Circulation. 2004;110:271-7.
28. Verhoeven AJ, Hilarius PM, Dekkers DW, et al. Prolonged
storage of red blood cells affects amino­ phospholipid
translocase activity. Vox Sang. 2006;91:244-51.
29. Vamvakas EC. Meta-analysis of randomized controlled trials
investigating the risk of postoperative infection in association
with whiteblood cell-containing allogeneic blood transfusion:
the effects of the type of transfused red blood cell product and
surgical setting. Transfus Med Rev. 2002;16:304-14.
Neurological ICU: Role of Red
Blood Cell Transfusion
BL Bhardwaj, Dharam Paul, Arun Bansal
Patients with severe traumatic brain injury (TBI) commonly
develop anemia. Anemia is one of the potential cause of
secondary injury, which may worsen neurological outcomes
for patients with neurological injury. Treatment of anemia
may include transfusions of packed red blood cells or
administration of erythropoietin. Erythropoietin treatment
of anemia after TBI has the additional potential of providing
neuroprotection. The neuroprotective mechanisms include
anti-inflammatory, antiapoptotic, and vascular actions.1,2
Transfusions of packed red blood cells restore hematocrit
and the carrying capacity of blood oxygen, but have been
associated with increased risk of infection, multiorgan failure
including respiratory failure, thromboembolic events, and
death. Studies have shown that for most critically ill patients,
there is no advantage of maintaining a higher hemoglobin
concentration.3-5 Still in critically ill patients, concern lingers
that hemoglobin concentrations as low as 7 g/dL may not
be tolerated in patients with severe TBI. Studies have either
shown no difference in mortality6 or suggested an association
between transfusion and a worse neurological outcome.6,7
Maintaining a hemoglobin concentration of approximately
10 g/dL has long been a management strategy to improve
cerebral oxygenation in patients with TBI. In studies of
patients with TBI and anemia, hemoglobin transfusion
does improve brain oxygenation in some patients.8,9 Other
potentially beneficial effects of maintaining a higher
hemoglobin concentration are to avoid increased intracranial
pressure induced by anemia, and to provide a higher blood
pressure and therefore better cerebral perfusion pressure.
This transfusion practice was expected to reduce neurological
injury, particularly during the acute recovery period when the
brain is most vulnerable to ischemic insults.10
Among patients with closed head injury, (Robertson
et al.)11 neither the administration of erythropoietin nor
maintaining hemoglobin concentration of at least 10 g/dL
resulted in improved neurological outcome at 6 months and
a greater incidence of thromboembolic events was observed
with this threshold. These findings do not support either
approach in patients with TBI.
7
Role of Red Blood Cell Transfusions
Red blood cell transfusion (RBCT) is a common therapy used
in the intensive care unit to treat anemia. However, due to
deleterious side effects and questionable efficacy, the clinical
benefit of RBCT in patients who are not actively bleeding is
unclear.12
Red blood cell transfusions (RBCTs) are intended to
improve tissue oxygenation. The notion that RBCT augments
tissue perfusion presumes that transfused blood efficiently
stores and off loads oxygen, and that compromised tissues
utilize the additional oxygen. However, studies have shown
that although transfusion increases oxygen carrying capacity
by augmenting hemoglobin concentration, it often fails to
increase oxygen utilization.13,14 The inability of transfused
blood to increase tissue perfusion has been attributed to a
series of biochemical and biomechanical changes that occur
during red blood cell (RBC) storage collectively termed the
“storage lesion.” The storage lesion results in decreased
oxygen delivery to tissues. After 7 days, stored blood is
depleted of 2,3-diphosphoglycerate, a compound that is
responsible for enhancing oxygen release from hemoglobin
to tissues15 which shifts the oxygen dissociation curve to the
left and reduces the amount of oxygen available for tissue
consumption. Furthermore, time-dependent changes in
stored blood lead to acidemia and hyperkalemia, which result
in RBC lysis and release of free hemoglobin,16 which is a nitric
oxide scavenger, therefore may result in vasoconstriction and
exacerbation of organ dysfunction.17
Structural changes, induced by RBC storage, have been
shown to compromise microvascular circulation. The storage
lesion also promotes increased RBC aggregability and
RBC endothelial cell adhesion, which may compromise or
obstruct microvascular circulation.18 Part of this effect may be
mediated by microparticles, a nucleoid membrane vesicles,
which increase in concentration with storage duration
Microparticles have been implicated in post-transfusion
thrombosis likely due to the expression of phosphatidylserine,
which promotes plasma-mediated thrombin generation.19,20
 Chapter 7  Neurological ICU: Role of Red Blood Cell Transfusion  25
Adverse Effects of Transfusion
Blood transfusion therapy is associated with adverse side
effects, including transmission of infections and immune
activation or immunosuppression. Both viral illness and
prion diseases may be transmitted by blood transfusion.
Risks of transmission of human immunodeficiency virus,
hepatitis C virus, and hepatitis B virus are 1 in 1,900,000; 1 in
600,000; and 1 in 220,000, respectively.21 Cytomegalovirus is
present in 4% of transfusions from healthy donors due to the
reactivation of latent cytomegalovirus in leukocytes.22
Transfusion modulates the immune system in 2
opposite ways: 1) it may heighten the immune response
(“alloimunization”), as in transfusion reactions, or it may
quell the immune response (“tolerance induction”), which
predisposes to nosocomial infections. Human leukocyte
antigens (HLA), specifically HLA-DR antigens, on donor
leukocytes partly determine which response ensues following
RBCT; shared HLA-DR antigens between donor and recipient
induce tolerance, whereas antigenic mismatch results in
immunization.23
Alloimmunization and consequent induction of HLA
antibodies and T-cell activation results in a number of clinical
syndromes, including transfusion reactions, transfusion
associated graft-versus-host disease, transfusion-related
acute lung injury (TRALI), and potentially the development
of various autoimmune diseases.23 TRALI is the number
1 cause of transfusion-related mortality.24,25 The most
common clinical features include: bilateral pulmonary
edema, hypoxemia, fever, dyspnea, and hypotension in the
presence of normal cardiac function.26 Plasma-rich blood
components (fresh frozen plasma and platelets) and high-
volume transfusion may predispose to TRALI.27
Tolerance induction after RBCT is associated with a
decrease in natural killer cell function, defective antigen
presentation, and a reduction in helper/suppressor
T-lymphocyte ratio, and has been linked to increased
predisposition to nosocomial and postoperative infections
and even to cancer recurrence. Transfusion was also
associated with an increased risk of multi-organ dysfunction
and acute respiratory distress syndrome.28-31
Clinical side effects due to the storage lesion result not
only from erythrocyte changes, but also from leukocyte
changes. Transfusion-transmitted infections are thought
to be due to contaminated leukocytes. Donor leukocytes
secrete cytokines, which have been associated with both
febrile nonhemolytic transfusion reactions (FNHTR) and
hemolytic transfusion reactions, in a time-dependent
manner after storage.32 Accumulation of lipid mediators,
capable of priming recipient neutrophils and exacerbating
multi-organ dysfunction, may result from leukocyte activity
on RBC membranes during storage.33 Leukocyte degradation
during storage results in release of oxygen free radicals
and proteases, which may also incite inflammation in the
transfusion recipient.34
Leukoreduction of stored blood might mitigate
the immunomodulatory effects of transfused RBCs,
leukoreduction reduce the rate of FNHTR. Universal
leukoreduction was associated with lower mortality, but not
a significant decrease in infection rate.35 Studies have failed
to demonstrate any beneficial effect of leukoreduction on
clinical outcome, including in-hospital mortality, ICU length
of stay (LOS), and antibiotic usage.36 Although the utility
and cost-effectiveness of universal leukoreduction remains
controversial, the majority of blood banking centers in the
United States has followed the lead of Canada by adopting
this practice.
Transfusion in the Neuro-ICU
Traumatic Brain Injury
Among patients with closed head injury, (Robertson et al.)11
neither the administration of erythropoietin nor maintaining
hemoglobin concentration of at least 10 g/dL resulted in
improved neurological outcome at 6 months and a greater
incidence of thromboembolic events was observed with this
threshold. These findings do not support either approach in
patients with TBI.
There are no prospective randomized studies of
transfusion in patients with TBI, and data are largely derived
from subgroup analyses of prospective studies in other
populations. Trials in general trauma patients suggest that
transfusion may increase the risk of both infection and
mortality. Dunne et al.37 demonstrated that transfusion of
more than 4 units of blood increases the risk of death by a
factor of 3 and increases the risk of perioperative infection
by a factor of 9.37 In another study, blood transfusion was
found to be an independent predictor of mortality, systemic
inflammatory response syndrome, ICU admission, and
increased ICU length of stay.30 Trauma patients who receive
blood transfusions have a two fold to sixfold increase in
systemic inflammatory response syndrome and a more
than fourfold increase in ICU admissions.30 In a sub study
of the TRICC trial, 203 trauma patients, 25% of whom were
identified as having TBI, were randomized to a liberal versus
restrictive strategy transfusion.38
Transfusion has been shown to improve PbtO2 in 4 studies
of severe TBI;39-42 however, the magnitude of augmentation
was small, the significance was questionable, and the
effect was inconsistent. Interestingly, in 1 of these studies,
RBCT resulted in a statistically significant increase in brain
oxygenation; however, 13 of 30 patients (43%) experienced a
decline in brain tissue oxygen tension (PbtO2) after RBCT.39 In
a study of 49 TBI patients with 564 episodes of compromised
PbtO2, blood transfusion improved compromised PbtO2
only 50% of the time.43 In contrast, interventions, such as
fraction of inspired oxygen (FiO2) augmentation, vasopressor
utilization, hyperosmolar therapy, and benzodiazepine
administration resulted in a >70% response rate.
26  Section 1  Clinical Hemotherapy
The variable response to RBCT observed in patients
with TBI may be due to the fact that secondary ischemia is
less common than previously thought. Physiologic studies
suggest that what was thought to represent ischemia
may actually signify mitochondrial failure.15 Cerebral
microdialysis has traditionally been used to identify regional
ischemia by sampling the interstitium for metabolites, such
as glucose, lactate, and pyruvate. The principal marker of
cerebral ischemia is the LPR. An LPR >40 has been correlated
with PET evidence of regional brain ischemia, particularly in
patients with SAH.44-46 However, an elevated LPR has also
been identified in TBI without evidence of ischemia; PET data
suggest that an elevated LPR corresponds to nonischemic
reductions in cerebral metabolic rate for oxygen, a measure
of mitochondrial function.46,47 Therefore, RBCT intended to
improve compromised blood flow may not be warranted.
Ischemic Stroke
While augmentation of oxygen carrying capacity (CaO2) and
oxygen delivery (DO2) might ameliorate symptoms of an
ischemic disease, concerns about viscosity-related reductions
in cerebral blood flow (CBF) have limited consideration of
RBCT in stroke. It has been postulated that increased viscosity
exacerbates stasis, compromising microcirculatory blood
flow to the ischemic penumbra. All port et al.48 demonstrated
that higher baseline hematocrit (>50%) was associated with
expansion of infarction and less reperfusion. This effect may
be more pronounced in women with hematocrit >50%.49
Moreover, elevated hematocrit levels have been associated
with carotid atherosclerosis,50 atrial fibrillation, unilateral
cerebral infarction, greater infarct size early mortality and
major disability after stroke.51,52
Other studies have noted a “U-” or “reverse J”-shaped
relationship between hematocrit and increased risk of stroke,53
suggesting that not only high hematocrit concentrations
(>50%), but also low hematocrit concentrations (<30%) are
also associated with an increased risk of ischemic stroke.
Reduced admission hemoglobin concentrations have been
associated with larger infarct volume and infarct expansion
on magnetic resonance imaging, and hemoglobin and
hematocrit nadir have been associated with poor outcome.54
Intracerebral Hemorrhage
A dose-dependent relationship between anemia and
intracerebral hemorrhage (ICH) volume may exist,55 the
effect of RBCT on outcome after ICH.56 Sheth et al.56 found
that RBC transfusion was associated with improved survival
at 30 days (p = 0.002) and decreased mortality at 30 days (
p = 0.02). Whether increased oxygen carrying capacity is
required in the post-ICH period during times of cerebral
edema, hydrocephalus, intracranial hypertension, or seizures
remains unknown. Secondary injury after ICH, character­
ized by iron-mediated neurotoxicity, macrophage activation,
and matrix metalloproteinase up regulation, may further
increase the demand for oxygen. Anemia may be associated
with increased hematoma volume, the primary predictor
of mortality in patients with ICH. Whether transfusion can
limit hematoma volume and improve outcome remains to be
determined.
Subarachnoid Hemorrhage
The impact of transfusion on subarachnoid hemorrhage
(SAH) outcome is largely unknown, and the relevant data are
mainly derived from retrospective clinical studies and from
physiologic studies. There exists relatively more data on the
relationship between anemia and outcome in patients with
SAH. Anemia consistently predicts, in a dose-dependent
fashion, infarction, death, and dependency57 and may
also predict cognitive impairment.58 Whether transfusion
mitigates these risks is unknown.
Red blood cell transfusion may be associated with
increased risk of cerebral vasospasm. In a retrospective study
of 441 SAH patients, postoperative RBCT was associated with
an increased risk of both angiographic and symptomatic
vasospasm, independent of smoking, Hunt-Hess grade,
Fisher group, and intraoperative rupture. It was postulated
that transfused blood, depleted of nitric oxide, might result in
a blunted vasodilatory response to vasospasm.
Other retrospective studies have demonstrated an
association between transfusion and increased frequency of
cerebral and extra-cerebral complications in patients with
SAH.59,60 Transfusion significantly increases the risk of major
(cardiac, pulmonary, renal, or hepatic) and minor (rash, deep
vein thrombosis) medical complications. In a study of 620
SAH patients, RBCT was identified as a risk factor associated
with the development of acute lung injury, independent of
severity of illness, clinical grade, and severe sepsis.61 RBCT
may be associated with an increased risk of infection and
inflammation; this could prove particularly deleterious
to the patient with SAH, as infection has been shown to
exacerbate delayed cerebral ischemia (DCI) and potentially
worsen outcome in SAH.62 Erythrocyte transfusion may
also be associated with an increased risk of thrombotic
events in SAH patients, independent of injury severity,
baseline demographics, comorbid conditions, other blood
component transfusion, and transcranial doppler ultrasound
(TCD) vasospasm.63 This may be explained by rheological
and storage-induced changes in RBC structure, coupled with
alterations in coagulation and fibrinolysis in SAH patients
that may result in an increased risk of thrombosis.
At present, a multidisciplinary consensus panel of the
Neurocritical Care Society strongly recommends that
patients should receive RBCT to maintain a hemoglobin
concentration >8 to 10 g/dL, based on moderate quality
data.64 A pilot study has shown that it is feasible to target
hemoglobin thresholds and to prospectively assess outcome
after transfusion; a prospective, randomized controlled trial
 Chapter 7  Neurological ICU: Role of Red Blood Cell Transfusion  27
to definitively determine transfusion thresholds in SAH
patients is required.65
Conclusion
It remains unclear whether RBCT to correct anemia in
brain injury patients who are not actively bleeding is
warranted. Although RBCT may augment brain oxygenation
in compromised tissue, it may also result in a paradoxical
decrease in perfusion. This variable effect may be the result
of prolonged blood storage. It is possible that patients with
impaired baseline perfusion benefit from RBCT more
than those without. Transfusion may be more effective in
patients with SAH, given the associated risk of DCI, whereas
the ischemia previously identified in TBI patients may
more accurately represent mitochondrial dysfunction less
responsive to transfusion. Individualized transfusion therapy
may be preferable, with use of physiological endpoints
instead of arbitrary hemoglobin levels. Large, prospective,
randomized controlled trials are needed to better define the
role of anemia, optimal hemoglobin threshold, and utility
of RBCT and are of paramount importance to improve the
management of patients in the neurological ICU.
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 Chapter 7  Neurological ICU: Role of Red Blood Cell Transfusion  29
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Blood Transfusion Alternatives
Harnoor Singh Bhardwaj, BL Bhardwaj, Aastha Miranpuri
There is no alternative developed as yet which has all the
properties of blood. The various alternatives to transfusion of
blood/blood products are:
• Pharmacological agents
• Recombinant hematopoietic growth factors
• Red cell substitutes
• Platelet substitutes.
Pharmacological Agents
The various pharmacological agents used to minimize blood
loss in bleeding patients are:
• DDAVP or desmopressin (1-deamino 8-D Arginine
Vasopressin)
• Epsilon aminocaproic acid (EACA)
• Danazol
• Aprotinin
• Intravenous immunoglobulin
• Vitamin K.
DDAVP or Desmopressin
It is synthetic analog of vasopressin. Desmopressin results in
the transient increase of factor VIII and von Willebrand factor
because of release of endogenous stores of these factors. It is
used as a hemostatic agent in patients with mild to moderate
hemophilia A and von Willebrand disease. DDAVP has also
been found useful in a wide variety of platelet functional
disorders including uremia, cirrhosis, drug induced
platelet dysfunction and myelodysplastic syndromes. It is
administered intravenously or subcutaneously as a single
injection. The effect is seen within 30 minutes, peaks at 90–
120 minutes and lasts for several hours. Doses are usually
repeated within 24 hours.
Epsilon Aminocaproic Acid
Antifibrinolytic activity. It inhibits the formation of plasmin by
blocking the binding of plasminogen to fibrin. EACA helps in
8
stabilizing clot and is used to enhance hemostasis in a number
of clinical conditions where primary fibrinolysis contributes
to bleeding. Accordingly EACA has been reported to be helpful
to control bleeding in patients with postpartum hemorrhage,
postprostatectomy hemorrhage and menorrhagia, but is
contraindicated in patients with disseminated intravascular
coagulation (DIC) where fibrinogen is necessary to maintain
organ function. The drug can be used locally or systemically.
Danazol
Danazol is a synthetic androgen. It has been used with
some success in few patients with chronic idiopathic
thrombocytopenic purpura (ITP) and may also stimulate an
increase in hematocrit in some patients with autoimmune
hemolytic anemia.
Aprotinin (Trasypol)
Aprotinin helps in decreasing the blood loss during surgery.
It has a protective effect on platelet function. It also inhibits
plasmin thus causes reduction in fibrinolysis which may
contribute to decreased blood loss.
Intravenous Immunoglobulins
Intravenous Immuno­globulins are used now-a-days to
transiently increase platelet count in children and adults
with ITP. The immunoglobulins most likely act by causing
reticuloendothelial cell (REC) blockade.
Vitamin K
It is a fat soluble vitamin required for the synthesis of vita-
min K dependant coagulation factors, i.e. factor VII, IX and
X. Body stores of vitamin K are limited and last only for two
weeks. Vitamin K deficiency can occur because of antibiotic
use, obstructive jaundice or due to fat malabsorption syn-
dromes.

Vitamin K depletion usually causes prolongation of PT
out of proportion to APTT because factor VII which has the
shortest half life out of vitamin K dependant factors has
little effect on APTT. Deficiency of vitamin K dependant
coagulation factors is best managed by treating the underlying
disease and by giving parenteral vitamin K, provided liver
function is adequate.
Recombinant Hematopoietic
Growth Factors
Recombinant hematopoietic growth factors are the synthetic
forms of naturally occurring molecules capable of stimulating
endogenous hematopoiesis.
Recombinant Human Erythropoietin
(R-HU EPO)
Erythropoietin (EPO) is produced by the kidney. It stimulates
red cell production by interacting with erythroid progenitor
cells via specific receptor site on erythroid colony forming
units (CFU-E). The purification of erythropoietin and the
development of recombinant human erythropoietin have
proved effective in correcting various chronic anemias,1 e.g.
• Anemia of chronic renal failure
• Anemia secondary to zidovudine (AZT) therapy in HIV
patients
• Anemia secondary to chronic disorders, e.g., connective
tissue disorder
• To facilitate autologous blood collection within predeposit
program.
Erythropoietin therapy has reduced considerably the need
of blood transfusion especially in patients of chronic renal
failure. Erythropoietin dose is 50–100 IU/Kg body weight
three times a week either in I/V or S/C (more effective).
Erythropoietin may have substantial role in reducing the
need for homologous blood transfusion by increasing the
red cell production prior to surgery. Contraindication of
erythropoietin is uncontrolled hypertension. All the patients
on erythropoietin therapy should have a hemoglobin level
measured at least once a week until suitable level is achieved
and periodically thereafter.
Other Blood Cell Growth Factors
Recombinant Granulocyte-colony stimulating factor (G-CSF)
and Granulocyte macrophage-colony stimulating factor
(GM-CSF) stimulate endogenous granulocyte production.
These are used for the treatment of chemotherapy induced
neutropenia as it decreases the duration of neutropenia and
also increases the tolerance to cytotoxic drugs. GM-CSF has
been approved for use in patients undergoing autologous
marrow transplantation, allogenic marrow transplantation or
in patients having bone marrow suppression due to anti-viral
agents. These growth factors are also used to stimulate and
Chapter 8  Blood Transfusion Alternatives  31
increase the yield of peripheral blood stem cells (PBSCs) from
peripheral blood stem cell donors. Side effect is bone pain.
Thrombopoietin Its clinical trials are in progress:
Recombinant Human Antihemophilic
Factor (Rh-Factor VIII)
It offers a good and safe alternative to human derived factor
VIII concentrate as it carries no risk of transmission of
hepatitis or AIDS.
Red Cell Substitutes
Although blood transfusion is effective, it is not without
risks. Allogenic blood transfusion may cause fatal hemolytic
reactions, transmit blood-borne infectious agents and
compromise overall immune function. It is therefore highly
desirable to have an artificial oxygen carrying fluid that is
readily available, free of infectious agents and can be used
independent of the recipient blood type.
Constitution of an Ideal Blood Substitute
Blood substitutes or synthetic blood are currently labeled as
“Oxygen carriers”. This is because they are unable to mimic
many of the other functions of blood; they do not contain
cells, antibodies, or coagulation factors. Their main function
is to replace lost blood volume and oxygen carrying capacity.
The ideal blood substitute could be defined by its increased
availability as compared to donated blood; oxygen carrying
capacity, equaling or surpassing that of biological blood;
volume expansion; universal compatibility: elimination of
cross matching; pathogen free: elimination of blood contained
infections; minimal side effects; survivability over a wider
range of storage temperatures; long shelf life and cost efficient.
Partial blood substitutes not involved in oxygenation are
platelet substitutes. They cover a different realm than oxygen
carriers and are created for mainly one purpose: coagulation.
They are considered mainly with the hopes of eliminating
risks of bacterial contamination and the short shelf life.
The blood oxygen carriers are divided into two categories:
1. Hemoglobin based oxygen carriers (HBOCs)
2. Perfluorocarbon emulsions (PFCEs).
Hemoglobin Based Oxygen Carriers
(HBOCs)
• Stroma free hemoglobin and cross linked hemoglobin
• Microencapsulated hemoglobin.
Stroma Free Hemoglobin and Crosslinked
Hemoglobin
Stroma free hemoglobin is prepared from outdated red blood
cells. In the solution hemoglobin dissociates into dimers and
32  Section 1  Clinical Hemotherapy
monomers.21 This hemoglobin is modified by pyridoxalation
which reduces its oxygen affinity and subsequent cross linking
with various biocompatible polymers increases its molecular
size so that it is not filtered by renal glomeruli. As yet there is
no report of the use of modified hemoglobin in human.
Microencapsulated Hemoglobin
The stroma free hemoglobin can be enclosed in artificial
membranes, made readily from phospholipids (synthetic
membranes).2 Hemoglobin in these artificial cells is no
longer exposed to extracellular environment. The synthetic
membrane is more stable than biological RBC membrane.
There are no blood group antigens and artificial cells do not
react with blood group antibodies. Although these cells have
proved effective in carrying oxygen in rats but they have very
short stay in circulation after intravenous injection. This
hemoglobin does hold promise as an emergency resuscitative
fluid.
Table 1 shows the comparison of HBOCs to transfused red
blood cells.1
Benefits
The general benefits of HBOCs over transfused red blood
cells are: No prior planning is needed, faster and better
O2 distribution, ready to use, no wastage, no equipment
is needed, long shelf life, no refrigeration, universally
compatible, no clerical errors, immediately offloads oxygen,
no 2,3-DPG, can be used by Jehovah’s witnesses.
Challenges
The challenges associated with the development of HBOC are:
• Availability—Development of HBOCs faces supply
challenge. While production of human Hb by recombinant
DNA could be a possible solution.2
Table 1: Comparison between hemoglobin based oxygen carriers and red blood cells
Infused hemoglobin based oxygen carriers
Onset of action Immediate
Oxygen affinity Red cell 2,3 DPG not required for oxygen release
Oxygen transport Red cells plus plasma
Risk of disease transmission Sterile pharmaceutical; no leukocyte exposure
Storage Room temperature; no loss of efficacy
Shelf life 36 months
Compatibility Universal
Preparation Ready to use
Viscosity Low
Duration of action Maximum of 3 days
• Immunological response—Immunological response to
the foreign protein remains a challenge.
• Short half-life—Current HBOc products have chemically
or genetically cross-linked Hb chains resulting in larger
molecules that are not readily filtered by the glomerulus,
thus possessing a greatly increased half-life.
• Increased oxygen affinity—Hb in the plasma has a much
higher affinity for O2 than it does within a red blood
cell. The increased affinity for O2 is due to lack of 2,3
diphosphoglycerate (DPG) in the plasma. Consequently,
the HbO2 dissociation curve shifts to the left, making such
a high affinity Hb not an ideal oxygen delivery substance.
However, chemically cross-linking the Hb structure has
the net effect of decreasing O2 affinity and optimizing
intracellular oxygen delivery.3
• Vasoactive properties—Vasoactive properties are one of
the major challenges facing the development of HBOCs.
Perfluorochemical Compounds
Perfluorochemicals (PFCs) are large organic compounds
in which all the hydrogen atoms have been replaced by
fluorine atoms. The high solubility of gases is a major reason
for the current biological use. For the use in blood substitute
preparations, liquid PFCs are preferred. Oxygen transported
by a perfluorocarbon is carried in a solution. It can dissolve
40–70% oxygen per unit volume, almost 3 times the oxygen
capacity of blood. They contain no antigens,4,5 do not carry
carbon monoxide and that is why could provide oxygen to
carbon monoxide victim until the patient is able to replace
the abnormal red cells.
PFCs, have a shelf life of 2 years and minimizes the risk
of infection or immunologic reaction from a transfusion. In
addition, PFCs dissolve oxygen (unlike hemoglobin, which
binds the molecule), which allow them to load/unload
oxygen 2 times faster than hemoglobin and extract >90% of
the transported oxygen.
Transfused red cells
2,3 DPG dependent
Red cell 2,3 DPG required for oxygen release
Red cells only
Risk minimized by improved donor selection;
leukocyte depletion
Refrigeration required; progressive loss of efficacy
42 days
Type-specific
Requires typing and cross-matching
High
Estimated 60–90 days

Another potential use relates to the PFCs small particle size,
which enables the suspension to penetrate occluded vessels in
situations such as cerebral ischemia or myocardial infarction.
PFCs have now been licensed for use in percutaneous
transluminal coronary angioplasty (PTCA). Perfluorocarbon
must be emulsified before administrating intravenously.
Since emulsified PFC particles have little osmotic and
oncotic pressure properties, electrolytes and plasma volume
expanders must be added to provide these needs.
In contrast, both the PFC and oncotic agent (hydroxyethyl
starch) are essential when the substitute is given to replace
blood lost by hemorrhage.
Disadvantages
• PFCs do not preferentially extract oxygen from the air as
haemoglobin does, so the oxygen level in a perfluorocarbon
solution equilibrates with oxygen level in the atmosphere.
Perfluorocarbon therapy therefore requires the concurrent
administration of 60–100% oxygen.4,5 A high oxygen
environment can render the patient vulnerable to oxygen
toxicity, especially when the concentration of oxygen
in inspired gas is above 10% for more than 24 hours.
Disadvantage is of oxygen toxicity.
• Second disadvantage of PFCs emulsion is their instability,
resulting in the need for frozen stage.
• A third disadvantage of perfluorocarbon is its retention in
the liver and spleen.
• The most extensively studied fluorocarbon is fluosol-DA
developed by Japanese. Fluosol contains two types of
perfluorocarbons in a mixture of balanced electrolytes,
lecithin and hydroxyethyl starch (HES).
Platelet Substitute
Platelet-derived Products
Frozen Platelets
Platelets can be cryopreserved in 6% dimethylsulfoxide
(DMSO) for up to 10 years when stored at -80°C. The
disadvantages are that they are cumbersome to use, much
more expensive to produce than fresh platelets, and the
freezing process appears to cause some morphological
defects. Cryopreserved platelets are the only alternative
to fresh platelets in clinical use, but their use is generally
limited to storing autologous platelets for transfusion in
alloimmunized patients with acute leukemia.
Cold Liquid-stored Platelets
There are three methods:
1. Inhibiting cytoskeleton actin assembly
2. Using thrombosol as a cryoprotectant
3. Using antifreeze glycoproteins
Chapter 8  Blood Transfusion Alternatives  33
Using inhibitors of cytoskeleton actin assembly prevents
the disc-to-sphere-shape change seen in chilled platelets.
Thrombosol has been reported to give greater retention of
in vitro function. Antifreeze glycoproteins are found in the
circulation of polar fish. The use of these glycoproteins in cold
storage of platelets has been shown to inhibit platelet shape
change and cold-induced activation, but no in vivo findings
have been reported.
Photochemically Treated Platelets
Photochemical treatment of liquid-stored platelets with
psoralen and long-wavelength ultraviolet radiation has been
explored as a means to inactivate bacteria, protozoa it acts by
disrupting their DNA. The photochemically treated platelets
have normal hemostatic function, but have slightly in vivo
recovery and survival.
Lyophilized Platelets
Lyophilization platelets are prepared using 1.8% paraformal-
dehyde and freeze-dried with 5% albumin. Rehydrated plate-
lets have a similar appearance to fresh platelets and have
most of the same surface proteins.6
Platelet-derived Microparticles
Platelet microparticles are microvesicles of platelet mem­
branes which are formed spontaneously during storage. The
microparticles have similar hemostatic properties as intact
platelets. They can be produced from outdated platelets.
Culture-derived Platelets
In 1995, the process of growing platelets in the laboratory
from megakaryocyte progenitors was described. The resulting
platelets appeared to be functionally and morphologically
similar to fresh donated platelets in vitro.
Synthetic Platelet Alternatives
Arg-Gly-Asp (RGD)-based Platelet Substitute
Fibrinogen is a molecule in the circulation that binds
activated platelets together. Specifically, it is the multiple
Arginine-Glycine-Aspartine (RGD) sequences in fibrinogen
which interact with surface proteins of platelets. Autologous
erythrocytes (red blood cells) can be prepared with fibrinogen
bound to the surface, or with just the RGD fragments bound to
the surface. These have been called “thromboerythrocytes”.
They have been shown to be hemostatically effective in some
animal models of thrombocytopenia.7
Fibrinogen-coated Albumin Microcapsules
Fibrinogen has also been used to coat microspheres or micro­
capsules of the human protein albumin. Two preparations
34  Section 1  Clinical Hemotherapy
have been evaluated in preclinical trials: Synthocytes
(developed by Andaris, UK) and Thrombospheres (developed
by Hemo­sphere). Thrombospheres have a smaller mean
diameter, and in vivo studies suggest that they may have a
longer duration of action.8
Liposome-based Agents
Two liposome-based agents have been studied: plateletsomes
and  factor Xa  with  phos­pholipid vesicles. Plateletsomes are
lipid vesicles with platelet glycoproteins on their surface. Both
have been shown to be hemostatically effective in vitro and in
some animal models, but the latter approach was associated
with high toxicity.9
Future Prospects
One hurdle to the development of new platelet products
and platelet substitutes is to define a way to quantify the
effects of platelets and related products on bleeding. Several
platelet substitutes look promising, but we will probably see
photochemically treated platelets and cold-stored platelets in
clinical use much sooner. Given the demand for platelets and
the extremely short shelf life of fresh platelets, there is real
incentive to produce a safe alternative.
References
1. Goodnough LT, Monk TG, Andriole GL. Erythropoietin therapy.
N Engl J Med. 1997;336:993-8.
2. Kumar R. Recombinant hemoglobins as blood substitutes:
a biotechnology perspective. Proc Soc Exp Biol Med. 1995;
208:150-8.
3. Reid TJ. “Hb-based oxygen carriers: are we there yet?”.
Transfusion 2003;43(2):280-7.
4. Chang TM. Future generations of red blood cell substitutes.
J Int Med. 2003;253(5):527-35.
5. Nazer CD. Review of clinical trails of oxygen therapeutics,
TATM 2000;51:27-31.
6. Bode AP, Read MS, Reddick RL. Activation and adherence
of lyophilized human platlets on canine vessel strips in the
Baumgartner perfusion chamber. J Lab Clin Med. 1999;133:
200-11.
7. Coller BS, Springer Kt, Beer JH, et al. Thromboerythrocytes: in
vitro studies of potential autologous, semi-artificial alternative
to platelet transfusion. J Clin Invest. 1992;89:546-55.
8. Davies Ar, Judge HM, May JA, Glenn JR, Heptinstall S.
Interactions of platelets with Synthocytes, a novel platelet
substitute. Platelets 2002;13:197-205.
9. Alving BM, Reid TJ, Fratantoni JC. Frozen platelets and platelet
substitutes in transfusion medicine. Transfusion 1997;37:866-
76.
Role of Whole Blood in
Transfusion Practice
Ashok Sharma
Historically, blood was  transfused  as whole blood without
further processing. Whole blood is obtained from human
donors by venesection and is collected into a sterile, dis­
posable plastic pack, which contains an anticoagulant-
preservative solution. This solution usually contains citrate,
phosphate, dextrose and adenine. There are variations in the
volume collected and in the type of anticoagulant used in
different countries. Blood collected in an anticoagulant can be
stored and transfused to a patient in an unmodified state. This
is known as ‘whole blood’ transfusion. However, blood can be
used more effectively if it is processed into components, such
as red cell concentrates, platelet concentrates, plasma and
cryoprecipitate. In this way, it can meet the needs of more
than one patient. Some blood banks have replaced this with
platelets collected by  plateletpheresis  because whole blood
platelets, sometimes called “random” platelets, must be
pooled from multiple donors to get enough for a therapeutic
dose.
The appropriate use of blood and blood products means
the transfusion of safe blood products only to treat a condition
leading to significant morbidity or mortality that cannot be
prevented or managed effectively by other means. Blood
transfusion can be a life-saving intervention. However, like
all treatments, it may result in acute or delayed complications
and carries the risk of transfusion-transmissible infections,
including HIV, hepatitis viruses, syphilis, malaria and Chagas
disease.
Blood is an expensive, scarce resource. Unnecessary
transfusions may cause a shortage of blood/components
for patients in real need. Intravascular transfusion of whole
blood has long been a well-recognized life saver during and
after major surgery and where there has been massive loss of
blood in an accident or in childbirth. Whole blood provides
red cells, stable clotting factors, and volume in each unit
that make it potentially beneficial in rapidly hemorrhaging
patients. Each unit (~450–500 mL) provides the equivalent of
one unit of RBCs and one unit of plasma. However, the plasma
may be deficient in labile clotting factors (V and VIII) since
9
these factors degrade rapidly during storage. The platelets
undergo conformational changes even with short-term
refrigerated storage that causes rapid removal of transfused
platelets by the reticuloendothelial system and thus are
unlikely to provide much improvement in hemostasis.
Around 92 million blood donations are collected annually
from all types of blood donors. The average annual collection
per blood center is 30000 in high-income countries, 7500 in
middle-income countries and 3700 in low-income countries.
Only 31% of the blood collected in low-income countries is
separated into blood components. The capacity to provide
patients with the different blood components when they
require, is thus still limited in these countries.
Indications of Whole Blood
Transfusion
Acute Hemorrhage
Whole blood can be transfused to replace red cells in acute
bleeding when there is also a need to correct hypovolemia. As
whole blood transfusion is limited to acutely hemorrhaging
individuals, dosing should be based on the patient’s clinical
condition, estimated blood loss, and other measures being
used to maintain hemodynamic stability. Such as seen
with major trauma, requiring massive transfusion and
rapid correction of anemia, coagulopathy, acidosis, and
hypothermia. Studies supporting this approach include
military trauma where they are able to transfuse very fresh
(<24 hours old) whole blood which is not currently routinely
available in civilian institutions.
Autologous Transfusion
Whole blood is no longer commonly available or used in most
of the United States. The most common use of whole blood
in the United States is currently autologous donations for
elective surgery.
36  Section 1  Clinical Hemotherapy
Exchange Transfusions
Reconstituted whole blood is used for neonatal exchange
transfusions, most commonly for hemolytic disease of the
newborn. It is sometimes used during pediatric cardiovascular
surgery as well as in neonatal hemodialysis. For dosing of
reconstituted whole blood for exchange transfusions, please
consult with your institutions blood bank medical director or
hematologist.
Cardiovascular Surgery or Hemodialysis
Whole blood is used during cardiovascular surgery or
hemodialysis, predefined dosing protocols should be setup
by the institution depending on type of procedure performed
and the cardiopulmonary circuits used at the institution.
Dengue Fever
In dengue fever, dengue hemorrhagic fever and dengue shock
syndrome management, blood transfusion is recommended
in cases with unstable vitals where hematocrit falls at the rate
of 10 mL/Kg/hr. Loss of blood—10% or more of total blood
volume. Preferably fresh whole blood to be used. Refractory
shock despite adequate fluid administration and declining
hematocrit. Replacement volume should be 10 mL/kg body
weight at a time and coagulogram should be done. If fluid
overload is present packed red cells are to be given.
World Health Organization (WHO) suggests to low income
countries to assess the clinical demand and feasibility of
component processing. Low income countries are hardly
capable of investing their resources in processing whole blood
into components, buying and maintaining material to process
plasma in 8–24 hour and keep at < – 25°C and monitoring and
managing quarantine and stock. In resource-limited settings,
blood is collected most commonly in whole blood units. The
World Health Organization (WHO) estimates that resource-
limited countries should begin to fulfil baseline clinical
demand if 10–20 whole blood units per 1,000 population are
collected each year.
As per Central drugs standard control organization, there
are a total of 2545 blood banks in India, out of which 1564
are private and 981 are owned by government. In Himachal
Pradesh, out of a total of 20 blood banks, 19 are government
owned. Out of these, blood separation units are present at
three centers, i.e. one each at Shimla, Tanda and Mandi. Thus
at most of the places, facility of blood component separation
is not available; hence most of the people are getting whole
blood for transfusion.
Institutional experience and national databases suggest
that a restrictive blood transfusion approach is being in­
creasingly implemented as best practice.
Suggested Reading
1. Christopher D Hillyer, Beth H Shaz, James C Zimring, Thomas
C. Abshire.  Transfusion Medicine and Hemostasis: Clinical
and Laboratory Aspects. Elsevier. pp.  45–.ISBN  978-0-12-
374432-6.
2. http://www.who.int/worldblooddonorday/media/who_
blood_safety_factsheet_2011
3. http://reference.medscape.com/drug/whole-blood-999509
4. http://www.cdsco.nic.in/forms/content
 Transfusion Practice in Surgery
Management of Preoperative
Anemia in Patients Undergoing
Elective Surgery
Manisha Shrivastava
Preoperative anemia is a very common finding in elective
surgical patients and remains undiagnosed till the patients
undergo preoperative diagnostic examination. Preoperative
anemia is associated with excess morbidity and mortality.
Correcting anemia by blood transfusion was a long standing
modality for such patients, however, understanding of
the complications associated with allogenic transfusions
such as infection, renal injury, and acute lung injury has
lead to adaptations of different strategies other than blood
transfusion. The data suggesting that morbidity and mortality
amongst patients receiving blood before or during surgery
is higher has also brought about reduction in the use of
allogenic blood to manage preoperative anemia.
Prevalence Depends on Many Factors
The reported prevalence of anemia in surgical patients varies
widely from 5–76% and depends on the patient’s disease and
co-morbidities, the surgical procedure and associated blood
loss, and the definition of anemia used. The prevalence of
preoperative anemia increases with patient age and is higher
in women than in men.1
Minimum Acceptable Hemoglobin
Concentration
The National Institute of Health Perioperative Consensus
Conference concluded that “otherwise healthy patients
with hemoglobin values of 10 g/dL or greater rarely require
preoperative or perioperative transfusion, whereas those
with acute anemia with resulting hemoglobin values of less
than 7 will frequently require red cell transfusions.” Whether
patients with hemoglobin values between 7 and 10 g/dL
should receive transfusions depends on the patient’s clinical
status.2
The minimum acceptable level of hemoglobin varies
for patients because individual persons vary in their ability
to tolerate and compensate for anemia. For example, at
rest, normovolemic young healthy persons with normal
10
cardiorespiratory function tolerate, at least briefly, a
hemoblobin concentration of 5 g/dL. Serum lactate and
oxygen consumption do not increase, and mild cognitive
impairment is reversible. However, this low level of
hemoglobin may not be benign to elderly patients with
cardiopulmonary disease. Many questions remain regarding
when we should transfuse red cells for acute anemia.3
Procedures that Usually Do versus Do Not
Require Transfusion
Surgical procedures that usually require transfusion include
coronary artery bypass graft, major vascular surgery, hip
replacement, major spine surgery with instrumentation,
hepatic resections, radical prostatectomy, and selected
neurologic procedures such as resection of arteriovenous
malformation. Surgical procedures that usually do not require
transfusion include cervical spine fusion, intervertebral
discectomy, mastectomy, hysterectomy, reduction mammo­
plasty, cholecystectomy, tonsillectomy, transurethral resec­
tion of the prostate, and vaginal and cesarean deliveries.4
Pathophysiology of preoperative, perioperative and post
operative anemia.
Perioperative anemia is a common clinical entity. It is
usually due to combination of various mechanisms, including:
pre-existing anemia prior to surgery; anemia due to impaired
erythropoiesis, including alterations of metabolism of iron and
erythropoietin (EPO); anemia due to increased destruction of
red blood cells (RBCs); and anemia due to iatrogenic causes.
Postoperatively, anemia resembles anemia of chronic disease
and is probably related to the effects of inflammatory mediators
released during and after surgery on the production and
survival of RBCs. Pro-inflammatory cytokines, such as tumor
necrosis factor, impair erythropoietin-dependent signalling
and iron homeostasis. Iatrogenic causes, notably excessive
phlebotomies, remain a major cause of perioperative anemia.
With increasing emphasis on restrictive blood transfusion
strategies, understanding these mechanisms is important for
the clinician.5
38  Section 1  Clinical Hemotherapy
Significance of Preoperative Anemia
Preoperative anemia is associated with adverse outcomes
after cardiac surgery. In a study outcomes after non-cardiac
surgery were studied with an aim to assess the effect of
preoperative anemia on 30-day postoperative morbidity and
mortality in patients undergoing major non-cardiac surgery.
The study concluded preoperative anemia, even to a mild
degree, is independently associated with an increased risk of
30-day morbidity and mortality in patients undergoing major
non-cardiac surgery.6
Recommendations for Detection,
Evaluation and Management of
Preoperative Anemia in Orthopedic
Surgery [Network for Advancement
of Transfusion Alternatives (NATA)]
A standardized approach for the detection, evaluation,
and management of anemia before surgery is of utmost
importance in the hospital settings of developing countries.
A multidisciplinary panel of physicians was convened by
the Network for Advancement of Transfusion Alternatives
(NATA) with the aim of developing practice guidelines for
the detection, evaluation, and management of preoperative
anemia in elective orthopedic surgery. A systematic literature
review and critical evaluation of the evidence was performed,
and recommendations were formulated according to the
method proposed by the grades of recommendation assess­
ment, development and evaluation (GRADE) working
group. It was recommended that elective surgical patients
have a hemoglobin (Hb) level determination 28 days before
the scheduled surgical procedure if possible (Grade 1C).
It was also suggested that the patient’s target Hb before
elective surgery be within the normal range, according to
Table 1: Management: blood delivery to operating room beginning with perceived need
Elective cases
Consider autologous and directed donor blood
Request blood bank to coordinate autologous, directed, and random
blood needed. Draw blood specimen from patient for blood bank,
before the day of surgery, if possible
Transport blood to operating room on the day of surgery Facilitate transport logistics if urgent, e.g. send person to blood
Check blood units and patient identity by two persons before
transfusion
Transfuse autologous before directed and directed before random units.
Monitor for adverse reactions
Return unused units to blood bank inventory
the World Health Organization criteria (Grade 2C). The
group also recommended that further laboratory testing to
evaluate anemia for nutritional deficiencies, chronic renal
insufficiency, and/or chronic inflammatory disease (Grade
1C). It was recommended that nutritional deficiencies be
treated (Grade 1C). The suggestion that erythropoiesis-
stimulating agents be used for anemic patients in whom
nutritional deficiencies have been ruled out, corrected,
or both (Grade 2A) and anemia should be viewed as a
serious and treatable medical condition, rather than simply
abnormal laboratory value. The authors concluded that the
implementation of anemia management in the elective
orthopedic surgery setting will improve patient outcomes.
Since the data suggested that the mortality and length of
stay are worsened with liberal use of transfusion, medical
alternatives to transfusion include iron supplementation
and erythropoiesis-stimulating agents (ESAs) could be
used to reduce the need for perioperative blood transfusion
compared with placebo, they are associated with an increased
risk of thrombotic events in surgical patients.7
Management of Preoperative Anemia
The indication to treat preoperative anemia depends on the
severity of the anemia, as well as the expected surgical blood
loss. For instance, if a patient has currently a hemoglobin
level of 9 g/dL and is expected to lose more than 500–1000
mL of blood, there is an increased likelihood of dropping the
hemoglobin to critical values (<7 g/dL) where transfusion is
indicated. In this group of patients, the hemoglobin should be
optimized, raising it by 2–3 g/dL preoperatively, to compensate
for the expected postoperative drop and attempt to prevent
the critical transfusion threshold.8,9 There is a definite link
between preoperative anemia in patients undergoing surgery
and perioperative needs of blood and the management plan
of elective and emergency cases has been depicted Table 1.
Emergency cases
Consider whether uncrossmatched blood
is indicated
Request to blood bank with patient’s blood specimen and
indicate urgency
bank to pick up blood
Check blood units and patient identity by two persons before
transfusion
Monitor for adverse reactions
Return unused units to blood bank inventory
 Chapter 10  Management of Preoperative Anemia in Patients Undergoing Elective Surgery  39
Blood management is a patient-centered standard of
care whereby strategies and techniques are used to reduce,
eliminate or optimize blood transfusions to improve patient
outcomes. It ensures safe, efficient use of the many resources
involved in the complex process of blood component therapy.
Its objectives are the translation of evidence into clinical
practice to minimize blood utilization, ensure consistency
among different physicians' practice behavior and
implement preoperative treatment focused on the etiology
of anemia as a “best practice” for elective surgical patients.
Perioperative blood management is poised at the confluence
of patient safety, resource utilization and system integration,
with consequent improvement in the overall quality of care.
Blood transfusion to correct anemia is currently considered a
marker of poor outcomes in medicine for multiple reasons. In
the current state of a decreasing donor pool, arbitrary use of
blood impedes effective resource allocation. It is associated
with serious complications (e.g. transfusion-related acute
lung injury, immune-modulation, transfusion-associated
circulatory overload), and there is substantial cost associated
with the blood banking process. In addition, recent studies
have shown that a blood-restrictive approach is associated
with improved outcomes in hip surgery patients.10
Conclusion
Anemia is a potent risk factor for mortality and morbidity
in surgical patients, and its management has begun to shift
away from allogenic blood transfusion in recent years. A
blood management program aimed at reducing allogenic
blood exposure for greater patient safety, hand in gloves with
the national anemia prevention program is the need of the
hour to reduce blood utilization and improve and prevent the
adverse outcomes; the program has achieved some reduction
in blood utilization in its first 7 months. Preoperative anemia
is associated with adverse outcomes after cardiac surgery but
outcomes after non-cardiac surgery are not well established.
References
1. Kumar A. Perioperative management of anemia: limits of
blood transfusion and alternatives to it. Cleve Clin J Med.
2009;76(Suppl 4):S112-8.
2. Consensus conference: Perioperative red cell transfusion.
National Institutes of Health. Conn Med. 1988;52(10):593-6.
3. Weiskopf RB, Viele MK, Feiner J, Kelley S, Lieberman J, Noorani
M, et al. Human cardiovascular and metabolic response to
acute, severe isovolemic anemia. JAMA. 1998;279(3):217-21.
4. NIH Expert Panel on Autologous Blood: Transfusion alert: Use
of autologous blood transfusion. 1995;335:703-13.
5. Singh S, Gudzenko V, Fink MP. Pathophysiology of perioperative
anaemia. Best Pract Res Clin Anaesthesiol. 2012;26(4):431-9.
6. Musallam KM, Tamim HM, Richards T, et al. Preoperative
anaemia and postoperative outcomes in non-cardiac surgery:
a retrospective cohort study. Lancet. 2011;378:1396-407.
7. Goodnough LT, Shander A, Spivak JL, et al. Detection,
evaluation, and management of anemia in the elective surgical
patient. Anesth Analg. 2005;101:1858-61.
8. Boucher BA, Hannon TJ. Blood management: a primer for
clinicians. Pharmacotherapy. 2007;27(10):1394-411.
9. Patel M, Auron M, Benitez-Santana SM, et al. Preoperative
blood management—paving the path to blood less surgery.
J Hosp Med. 2011;6(Suppl 2):S124.
10. Carson JL, Terrin ML, Noveck H, et al. Liberal or restrictive
transfusion in high-risk patients after hip surgery. N Engl J
Med. 2011;365:2453-62.
Blood Conservation Strategies

DP Singh
11
INTRODUCTION
Blood conservation is a collaborative, advanced medical
approach to patient care that reduces the need for donor
blood transfusions or blood components. Blood conservation
is important in surgery because of risk associated with blood
transfusion and due to shortage of blood supply. Potential
health benefits include faster recovery reduced hospital
stays, reduced risk of infections and reduction in donor blood
transfusions.1

METHODS
With the arrival of minimal invasive surgery, robotic surgery
and new vessel sealing instruments it is possible to perform
bloodless surgery. Blood conservation measures include2–4:
• Perioperative Figure 1  Harmonic scalpel
• Intraoperative
• Postoperative.
Blood conservation strategies start from perioperative
period which include:
• Anemia correction
• Identify and manage bleeding risk
• Manage anticoagulation carefully
• Procedure planning and timing surgery
• Autologous blood donation.
Intraoperative methods:
• Meticulous hemostasis
• Blood sparing surgical techniques
• Permissive hypotension
• Autologous blood options
• Hemostatic agents.
There are various devices available for obtaining meticu-
lous hemostasis which include:
• Harmonic scalpel (Fig. 1)
Figure 2  Ligasure
• Ligasure (Fig. 2)

Figure 3  CUSA
• CUSA—used in neurosurgery, liver surgery (Fig. 3)
• Robotic arms which provide 7 degrees of freedom (Fig. 4)
Postoperative care includes:
• Avoiding secondary hemorrhage
• Control of infection
• Maintain normothermia.
CONCLUSION
Optimal patient blood management is the key to success to
conserve this scarce, expensive and potentially infectious
resource. With our current understanding of pathophysiology
of blood transfusion, cell savers, autologous blood transfusion
and that we had learned from damage control resuscitation in
warfare and with the invention of modern minimal invasive
Chapter 11  Blood Conservation Strategies  41
Figure 4  Robotic surgery
and robotic surgical techniques it is necessary to come
together and share our experience with each other as team
for welfare of our patients and humanity so that they can get
maximum benefit.
REFERENCES
1. LD Petz., et al’s Clinical Practice of Transfusion Medicine, 3rd
edn.
2. Haemostasis and Thrombosis Task Force (for the British
Committee for Standards in Haematology). Guidelines on oral
anticoagulation: third edition. British J Haem. 1998;101:374-87.
3. Guidelines for Autologous Transfusion part II: peri-operative
haemodilution and cell salvage. Br J Anaes. 1997;6:768-71.
4. Page C, Retter A and Wyncoll D Blood conservation devices in
critical care: a narrative review. Annals of Intensive Care. 2013;
3:14.
Thromboelastography
Anupam Verma
To assess the viscoelastic properties of blood there are
currently various coagulation monitoring devices in the
market namely Thromboelastograph (TEG) (Hemoscope
Corporation, USA), ROTEM (Pentapharm GmbH, Germany),
and Sonoclot analyzer Sienco Inc., USA. These coagulation
analyzers provide graphical representations of the dynamics
of fibrin polymerization in citrated or noncitrated whole
blood, platelet-rich or platelet-poor plasma, with or without
activation.
The paradigm shift in our understanding about classical
coagulation cascade occurred due to introduction of a cell-
based model which emphasizes the importance of tissue factor
as the initiator of the coagulation cascade and the pivotal role
of platelets for intact hemostasis. This new understanding
explains the poor correlation between traditional tests of
coagulation and clinical bleeding and has generated renewed
interest in viscoelastic tests for diagnosing derangement in
hemostasis and to guide transfusion therapy.
TEG assesses the viscoelastic properties of blood samples
under low shear conditions. It measures the clot’s physical
property by using a stationary cylindrical cup that holds the
blood sample and oscillates through an angle of 4°45 with
each rotation cycle lasting 10 sec. The TEG tracing shows
complete dynamics of clot formation and lysis over time
(Fig. 1). The main TEG parameters are defined as under.
Figure 1  TEG on citrated blood sample in a patient of DIC
12
R value is time from the beginning of the trace until
amplitude of 2 mm is reached. This denotes the time taken
from the placement of the blood sample in the cuvette to
the initial fibrin formation. It represents a deficiency in
coagulation factors, effects of endogenous heparin, and/or
hemodilution. The treatment with FFP or no treatment would
depend on the clinical picture. Conversely, a shortening
in the R value (<2 min) would indicate a hypercoagulable
picture and treatment with an anticoagulant of choice may
prove beneficial.; K is the time from the end of R until a fixed
level of clot strength is reached, i.e. amplitude of the trace
is 20 mm. A low value may indicate the need for fibrinogen
or cryoprecipitate administration, and a high value may
indicate hypercoagulability for which the patient may benefit
from anticoagulant therapy; α-angle (clot formation rate)— It
is the angle of the trace from the horizontal at a point on the
trace until amplitude is 20 mm. It measures the rate of fibrin
build up and cross linking and to some extent platelets also
contribute to α angle. Both K and α angle measure speed of
clot formation or clot kinetics; MA (maximum amplitude)—It
is a measure of maximum strength of the clot and assesses the
function of the platelets and to some extent fibrinogen also;
A low value can result from hypofibrinogenemia, decreased
platelet function, or quantity. Transfusing platelets may
improve hemostatic condition. An MA >69 is an evidence
of a prothrombotic state, and an anticoagulant of choice
may be clinically indicated. CI (coagulation index). The
patient’s overall coagulation profile is calculated from the R,
K, MA and angle. The qualitative analysis thus obtained from
TEG can facilitate administration of right blood product or
anticoagulant in correct amount. The TEG assay can also be
used for preoperative risk stratification based on individual
hemostatic conditions.
Besides standard TEG, Platelet Mapping which is an
extension of TEG technology, in addition to providing
information on clot formation and lysis of whole blood
samples, it quantifies the contribution of fibrin, adenosine
diphosphate (ADP) receptor and thromboxane A2 (Tx A2)
receptor in clot strength.1 The TEG Platelet Mapping assay

enables relating the percent platelet inhibition to the
individual’s maximum uninhibited platelet function. This
information allows monitoring of effectiveness of anti-
platelet agents, aspirin and clopidogrel, via inhibition of
TxA2 and ADP receptors. This investigative tool is important
where we need to monitor the effects of antiplatelet drugs
especially in patients from cardiology, cardiac surgery,
Neurology, Interventional radiology. The existence of a high
level of individual variability in platelet responsiveness to
antiplatelet therapy and on-treatment platelet reactivity have
been confirmed in the majority of clinical studies examining
antiplatelet therapy efficacy in patients undergoing
percutaneous coronary intervention. Platelet mapping can
also depict whether a delay in surgery is necessary or, may
possibly proceed earlier than anticipated.
Various studies suggest that TEG could be a complimentary
test to current standard coagulation assays and in some
cases, a superior method altogether. In theory, TEG has
many of the properties of an ideal coagulation test. However,
there are issues with this technology as well. First, TEG was
designed for use with fresh whole blood samples, but use of
fresh whole blood requires initiation of the test within 4–6
minutes of sample collection which is not always possible.
Thus citrated blood samples were used for laboratory based
testing. However, both systematic differences and artifacts
in TEG tracings have been described when comparing fresh
whole blood to citrated blood samples.2,3 Differences in
patient populations, use of clot activators, and other factors
make the studies difficult to directly compare. Beyond
choice of sample types and issues of timing between sample
collection and testing, TEG and other viscoelastic tests are
subject to unique pre-analytical and analytical artifacts
that can significantly affect the results. The manufacturer’s
manual provides normal reference values for most but not all
different techniques. It is recommended that one should have
local reference values for TEG which is not very practical.4
Besides it cannot detect the disorders of primary hemostasis
and not sensitive to mild coagulation factor deficiencies.
Chapter 12  Thromboelastography  43
Variations in hematocrit are known to affect the results
with increased and decreased hematocrit leading to TEG
picture that indicate hypocoagulable and hypercoagulable
states, respectively. Thromboelastographic results need
to be carefully interpreted in presence of severe anemia,
thrombocytopenia and hemodilution as these conditions can
variably affect the test results.5 TEG has still not been adopted
in mainstream clinical practice due to poor standardization
and validation and artifacts it can introduce if not performed
correctly as it is not fully automated. Bubbles can appear in
the cup if pipetting technique is not good which can affect the
test result.
As this technology diversifies with different reagents and
applications, it is important to understand these differences
when interpreting any data generated. Overall, TEG is a
powerful tool, but its implementation requires thought and
planning to insure that the test is performed correctly and
results are interpreted cautiously taking into consideration
the clinical picture and results of other laboratory tests.
References
1. Agarwal S, Coakley M, Reddy K, Riddell A, Mallett S.
Quantifying the effect of antiplatelet therapy: A comparison
of the platelet function analyzer (PFA-100) and modified
thromboelastography (mTEG) with light transmission platelet
aggregometry. Anesthesiology. 2006;105:676-83.
2. Camenzind V, Bombeli T, Seifert B, et al. Citrate storage affects
thromboelastograph analysis. Anesthesiology. 2000;92:1242-9.
3. Johansson PI, Bochsen L, Andersen S, Viuff D. Investigation
of the effect of kaolin and tissue factor-activated citrated
whole blood, on clot forming variables, as evaluated by
thromboelastography. Transfusion. 2008;48:2377-83.
4. Scarpelini S, Rhind SG, Nascimento B, et al. Normal range
values for thromboelastography in healthy adult volunteers.
Braz J Med Biol Res. 2009;42:1210-17.
5. Bolliger D, Seeberger MD, Tanaka KA. Principles and practice
of thromboelastography in clinical coagulation management
and transfusion practice. Transfusion Medicine Reviews. 2012;
26:1-13.
Transfusion Support
in Cardiac Surgery
Rajendra K Chaudhary
Cardiac surgery, above all other major surgical procedures,
continues to place the largest demand on the available blood
supply. Despite major advances in perioperative blood
conservation, transfusion rates in cardiac surgery remain
high with large variations among individual centers. Blood
transfusion is commonly used in cardiac surgical patients
and accounts for about 10–20% of blood transfused. It is
noted that approximately 10–20% of patients consume 80% of
total blood transfused among cardiac surgery patients.
Transfusion variability in
Cardiac Surgery
In spite of published guidelines there are tremendous
variations in transfusion practices among physicians and
institutions. The incidence of blood transfusion among
patients undergoing cardiac surgery has been reported to vary
between 27 and 92%. The observed variability in transfusion
practices may be reduced by altering blood use in this high
usage group. These wide differences in transfusion rates
may be explained by a variety of reasons including different
patient populations, differences in procedure-related factors,
traditions, and norms. Therefore, individual centers should
pay attention to their current transfusion practice.
Potential benefits of red blood cell
transfusion in cardiac surgery
Allogeneic blood transfusion may be given to treat life-
threatening hemorrhage during surgery, but most often
transfusion of blood components is given to increase
the oxygen-carrying capacity of the blood or to improve
hemostasis. There is, however, a lack of clear evidence
regarding the benefit of RBC transfusion when it comes
to enhancing the oxygen-carrying capacity of blood.
Only a few randomized trials were carried out to identify
the potential benefits of RBC transfusion. Randomized,
controlled study in 838 critically ill, normovolemic patients
in an intensive care unit (ICU) produced equivalent results
13
when the rates of all-cause mortality at 30 days and severity
of organ dysfunction when a restrictive strategy of red-cell
transfusion, i.e. hemoglobin concentration between 7 and
9 g/dL, was compared with a liberal strategy, i.e. hemoglobin
concentration at 10–12 g/dL. The restrictive strategy of RBC
transfusion was at least as effective as and possibly superior
to the liberal transfusion strategy. The restrictive strategy also
appeared to be safe in critically ill patients with cardiovascular
disease, with the possible exception of patients with acute
myocardial infarction and unstable angina.
Another randomized study in 428 elective, primary
coronary artery bypass grafting (CABG) patients found that
there were no differences in clinical outcomes, including
morbidity and mortality, between two groups of patients
assigned to receive RBC transfusion if the postoperative
hemoglobin was <8 g/dL or <9 g/dL, respectively. A
number of database studies in cardiac surgery reported that
decreased perioperative hematocrit values is associated with
decreased survival. In a study in 6,980 patients undergoing
CABG, the lowest hematocrit during cardiopulmonary
bypass (CPB) was significantly associated with increased risk
of in-hospital mortality, intra- or postoperative placement
of an intra-aortic balloon pump, and return to CPB after
attempted separation.
No “optimal” hematocrit or hemoglobin concentration
is established in cardiac surgery. The classical “10/30” rule,
suggesting that transfusion should be given to maintain a
hemoglobin concentration of 10 g/dL and a hematocrit of
30%, was abandoned several years ago. Today, much lower
hemoglobin and hematocrit values are accepted. However,
there are no clinically available methods for measuring
regional tissue oxygenation to identify when there is a need
for RBC transfusion. Observational studies among CABG
patients, who refused blood transfusions for religious
reasons, suggest that survival without transfusion is possible
at low hemoglobin concentrations.
During the past three decades, traditional transfusion
triggers were constantly challenged. Despite published
blood transfusion guidelines and reviews on transfusion
 Chapter 13  Transfusion Support in Cardiac Surgery  45

management in patients undergoing cardiac surgery there is

still no consensus as to when transfusion is needed.

Predictors of allogeneic
red blood cell transfusion
during Cardiac Surgery
Knowledge about predictors of bleeding and transfusion
is a prerequisite for preoperative risk stratification and
use of rational blood conservation techniques. Not all
patients are at the same risk for transfusion requirement.
Some patient variables can be used to predict the risk for
perioperative transfusion. Need for packed red blood cell
(PRBC) transfusion in isolated, first-time CABG patients
can be predicted preoperatively. The strongest predictors
are use of CPB, hematocrit ≤ 40%, body weight ≤ 70 kg, and
serum creatinine > 100 μmol/L. Female gender, older age,
higher Euro score, and number of distal anastomoses are
also significant predictors. Ability to identify patients at
risk of PRBC transfusion would save blood bank efforts and Figure 1  Mechanism of bleeding during cardiopulmonary bypass
resources and allow the employment of a targeted blood
conservation policy in CABG patients.
Recently, an externally validated transfusion risk under­ an important and delicate balance between anticoagulation
standing scoring tool (TRUST) for assessing this risk in adult during CPB and proper hemostasis after CPB. These
patients undergoing cardiac surgery has been proposed patients are at risk for excessive perioperative blood loss
(Table 1). often requiring transfusion of blood products. In the past,
clinicians administered blood products empirically due to
long turn around times of laboratory-based coagulation tests.
Mechanism of Bleeding in Patients
This practice exposes patients to inappropriate blood therapy
Undergoing Cardio Pulmonary Bypass resulting in increased morbidity and mortality and hospital
Bleeding during cardio pulmonary bypass is due to various costs. Today, point-of-care (POC) devices are available
factors as shown in the Figure 1. providing rapid bedside monitoring to aid the clinician in
directing appropriate targeted therapy. The majority of POC
devices used today can perform multiple coagulation tests.
Point-of-Care (POC) Tests in Cardiac Surgery The use of transfusion algorithms in conjunction with POC
The hemostatic management of patients undergoing cardiac testing has been shown to reduce both transfusion require­
surgery with cardiopulmonary bypass (CPB) necessitates ments and blood loss in cardiac surgery.

Table 1: Transfusion risk scoring system

TRUST Transfusion 2006;46:1120 (all cardiac surgeries) Magovern et al. Ann Thorac Surg. 1996;61:27 (CABG only)
Factor Score Factor Score
Hb < 13.5 g/dL 1 Lower red cell mass 2
Weight < 77 Kg 1 Low body mass index 2
Female gender 1 Female gender 2
Age > 65 years 1 Age > 74 years 2
Emergency surgery 1 Emergency surgery 4
Creatinine > 1.3 mg/dL Creatinine > 1.8 mg/dL 1
Previous cardiac surgery 1 Previous cardiac surgery 1
Nonisolated operation 1 Diabetes 1
Left ventricle EF < 0.3 2
Albumin < 4 g/dL 1
46  Section 1  Clinical Hemotherapy
Point-of-care of platelet function tests
•  Platelet function analyzer (PFA – 100)
•  Verify now
•  Platelet works
Viscoelastic tests of clot formation
•  Sonoclot
•  Thromboelastography
•  Rotational thromboelastometry
•  Impact cone and plate analyzer
Point-of-care testing of heparin effect
•  Activated clotting time
Adverse Outcomes following Allogeneic Red Blood
Cell Transfusion in Cardiac Surgery
Potential immunological and nonimmunological risks related
to allogeneic transfusion are well described. An increasing
number of observational studies indicate that allogeneic RBC
transfusion during CABG surgery is associated with both early
and long-term mortality, but conflicting results were found
among other patient groups. Transfusions seem to have no
effect on the long-term mortality rate after isolated valve
surgery; however, transfusion is associated with increased
mortality when valve surgery is combined with CABG. Studies
among patients undergoing surgery for hip fractures showed
both increased and no association between transfusion and
long-term mortality, respectively. In a study in ICU patients,
transfusions were associated with improved long-term
survival. These findings suggest that the type of surgery may
play a role in the outcome following transfusion.
Allogeneic RBC transfusion following cardiac surgery,
including CABG, was also reported to be associated with
postoperative morbidity, as shown in Table 2. A number
of observational studies indicated an association between
Table 2: Postoperative morbidity associated with allogeneic red blood cell transfusion in cardiac surgery
Studies Number of patients
Michalopoulos et al. 1998. Eur J Surgery 2,615
Leal-Noval et al. Chest. 2001;119:1461 738
Chelemer et al. Ann Thorac Surg. 2002;73:138 533
Koch et al. Crit Care Med. 2006;34:1608 11,963
Rogers et al. Am Heart J. 2006;152:1028 8,518
Scott et al. Ann Card Anaesth. 2008;11:15 1,746
Koch et al. Ann Thorac Surg. 2009;88:1410 16,847
storage time of RBC and patient outcome in cardiac surgery.
However, this association was not confirmed by other
studies, and the strength and causality of this association
remain uncertain. These differences may be attributed to the
heterogeneity of patient populations, transfusion of different
blood products, different outcome measures, and different
study designs.
Blood Conservation during Cardiac Surgery
Blood conservation during cardiac surgery is used to reduce
bleeding and allogeneic blood transfusion. Available tech­
niques include screening for risk factors associated with
bleeding, use of drugs to increase the preoperative blood
volume or to decrease postoperative bleeding, devices
that conserve blood (e.g. intraoperative cell saving and
postoperative reinfusion of shed mediastinal blood), inter­
ventions that protect the patients’ own blood from the
stress of the operation, and from CPB (e.g. intraoperative
normovolemic hemodilution). Most important is a multi­
modal approach because of the multifactorial causes of
bleeding. Among frequently used techniques for blood
conservation are pharmacological strategies. Antifibrinolytic
drugs have been used for almost two decades. Two drugs
were used equally frequently; the synthetic lysine derivate
tranexamic acid (TA), and the bovine-derived aprotinin. TA
acts mostly by competitive inhibition of the lysine binding
sites in plasminogen, resulting in retardation of fibrinolysis.
Aprotinin, a serine protease inhibitor, mostly acts by inhibiting
plasmin, resulting in attenuation of fibrinolysis. Both drugs
are effective in reducing postoperative bleeding and the
need for blood transfusion. However, an observational study
in 2006 reported an association between aprotinin and an
increased risk of renal failure, myocardial infarction, heart
failure, and stroke. In addition, aprotinin may increase the
risk of saphenous vein graft occlusion in CABG. Aprotinin
was, therefore, withdrawn from the market in November
2007.
Outcome associated with transfusion
Severe sepsis
Nosocomial pneumonia
Respiratory and surgical site infection
Renal failure, prolonged ventilatory support, serious infection,
neurologic events
Infection, pulmonary dysfunction
Increased time to extubation, stay in the ICU, and postoperative
length of stay
Pulmonary morbidity
 Chapter 13  Transfusion Support in Cardiac Surgery  47
SUGGESTED READING
1. Alghamdi AA, Davis A, Brister S, et al. Development and
Validation of Transfusion Risk Understanding Scoring Tool
(TRUST) to stratify cardiac surgery patients according to
their blood transfusion needs. Transfusion. 2006;46:1120-9.
2. Chelemer SB, Prato BS, Cox PM, et al. Association of bacterial
infection and red blood cell transfusion after coronary artery
bypass surgery. Ann Thorac Surg. 2002;73:138-42.
3. Koch C, Li L, Figueroa P, Mihaljevic T, Svensson L, Blackstone
EH. Transfusion and pulmonary morbidity after cardiac
surgery. Ann Thorac Surg. 2009;88:1410-8.
4. Koch CG, Li L, Duncan AI, et al. Morbidity and mortality
risk associated with red blood cell and blood-component
transfusion in isolated coronary artery bypass grafting. Crit
Care Med. 2006;34:1608-1.
5. Leal-Noval SR, Rincon-Ferrari MD, Garcia-Curiel A, et al.
Transfusion of blood components and postoperative infec­
tion in patients undergoing cardiac surgery. Chest. 2001;119:
1461-8.
6. Magovern JA, Sakert T, Benckart DH, Burkholder JA, Liebler
GA, Magovern GJ Sr, et al. A model for predicting transfusion
after coronary artery bypass grafting. Ann Thorac Surg. 1996;
61:27-32.
7. Michalopoulos A, Stavridis G, Geroulanos S. Severe sepsis in
cardiac surgical procedures. Eur J Surg. 1998;164:217-22.
8. Rogers MA, Blumberg N, Saint SK, Kim C, Nallamothu BK,
Langa KM. Allogeneic blood transfusions explain increased
mortality in women after coronary artery bypass graft surgery.
Am Heart J. 2006;152:1028-34.
9. Scott BH, Seifert FC, Grimson R. Blood transfusion is associated
with increased resource utilization, morbidity and mortality in
cardiac surgery. Ann Card Anaesth. 2008;11:15-9.
Transfusion Practice
in Arthroplasty
Manuj Wadhwa, Kunal R Patel
INTRODUCTION
Orthopedic surgery can be divided into the fields of elective
orthopedics and trauma. The elective aspects, particularly
joint replacement and spinal surgery, are well suited to the
many techniques available to help reduce the use of allogenic
blood.
Today over 30,000 joint replacements take place in India
annually, and the numbers are exponentially rising with each
passing year. The projected rise to 1,00,000 joint replacements
annually by 2020 in India puts into perspective the need of
blood salvage practices.
The problems of allogenic transfusion are already well
documented both in the reduced availability of blood and the
multitude of potential risks with its use.
The amount of blood loss is increased in cases of revision
hip and knee arthroplasty cases due to multiple factors like
tourniquetless surgeries in infected knees, metaphyseal bone
loss leading cancellous bone bleeding, fibrosis of previous
surgery scar leading to retraction of bleeding small vessels,
staged surgeries in infected joints.
Surprisingly, it is only recently that useful data has been
collected on a large scale.1 This data allows us to see the scale
of the problem. 330 Orthopedic surgeons across the USA
combined the information of blood management for patients
having hip or knee arthroplasty. In the USA, the standard of
care is usually to offer autologous predonation. A transfusion
(autologous or allogenic) was given to 46% of the 9,482
patients. The prevalence was higher in hip arthroplasty (57%)
than knee arthroplasty (39%). Despite the high collection rate
of autologous blood, only 55% of these units were actually
given back to the patient. Unilateral knee arthroplasties,
primary and revision, were associated with the most wastage.
Breakthrough transfusions of allogenic blood in patients who
had predonated blood were necessary in 9% of patients. As
expected this was most likely with revision hip arthroplasty.
In formulating a strategy for blood management, it is
important to identify the patients most at risk. This allows
the treatment to be tailored to the individual. Not only is
14
this beneficial in the clinical setting but also in providing
the most cost-effective solutions. It seems rather simplistic
to state that a patient with a lower preoperative Hb is more
likely to need a transfusion. Nevertheless, many studies have
detailed the likelihood of transfusion based upon the patients
preoperative status.1-5 Most institutions use the maximal
surgical blood order schedule (MSBOS). This certainly
improves the efficiency of blood ordering practices but only
deals with the group as a whole, not the individual.
Estimated blood loss, age, weight and aspirin use are all
indicators but by far the strongest predictor of the need for
transfusion is the preoperative Hb level. Whenever this falls
in the range of 8–10 g/dL the patient has a significantly higher
risk of requiring allogenic blood.
RECENT STUDIES AND RECOMMENDATIONS
FOR SPECIFIC OrthopeDIC PROBLEMS
Primary Total Hip Replacement
Total hip arthroplasty (THA) is one of the most common
elective procedures in orthopedics. Current data suggests
over half of these patients will receive a transfusion of
allogenic or autologous blood. The true blood loss is hard
to accurately assess and this causes problems when trying
to compare reports from different sources. The measured
loss suction, sponge weighing and drainage is invariably
much lower than the true loss if this is calculated using the
pre- and postoperative Hb levels. The true loss is variable
and depends upon both the patient and the surgeon with an
average around 1,600 mL. Overall, 53% of patients received a
transfusion, on average 2 units.
The majority of patients participate in a predonation
programme (65%). In the remainder, with no predonated
blood, 32% received an allogenic transfusion compared to a
16% breakthrough rate in the predonation group. There is also
a great increase in the transfusion rate when preoperative
anemia is present.

A predonation program obviously does decrease the need for
allogenic blood in primary THA. However, there are still some
serious concerns:
• Autotransfusion is not completely safe. Clinical errors and
infection can still occur.
• There is a high level of wastage. About half the units are not
used.
• Preoperative anemia is more likely with predonation. The
erythropoietic response to phlebotomy is unexpectedly
rather poor.
• Predonation is neither cheap nor easy to arrange.
• The use of hypotensive epidural anesthesia has been
shown to reduce the blood loss significantly and is used in
many orthopedic centers routinely.6
• Acute hemodilution is safe and effective but probably not
required for the majority of primary THAs. It requires a
high level of skill and is time consuming.
• There is some debate concerning the effect of different
implants. 25 matched pairs of THAs, half uncemented
and half hybrid or cemented, were compared. There
was no significant difference noted in the blood loss and
transfusion rate.7
• Intraoperative salvage is costly in time and staffing and
rarely warranted in primary THA. Postoperative reinfusion
drains can be used but most blood loss is intraoperative
and the trend is now to avoid the routine use of drains.8
Revision Hip Replacement
Templating and rehearsal of the procedure and familiarity
with the instruments and implants are vital. Revision surgery
is accounting for an increasing percentage of hip arthro­
plasty. The implants inserted in the 70s and 80s are now
reaching 15–20 year follow-up and the failure rate is rising
significantly. Revision surgery is much less predictable than
primary surgery and preoperative planning is more difficult.
At times the surgery is no worse than a primary procedure,
but more likely there will be problems with dense scar
tissue, large areas of exposed bone, osteolysis and fractures
and a greatly increased operative time. All this adds up
to increased blood loss, typically 50–100% greater than a
primary procedure. Typically 3 units of blood are transfused.
Breakthrough allogenic transfusion with a predonation
program is highest with revision hip surgery at 21% in one
series.1
Primary Total Knee Arthroplasty
In 100 consecutive primary total knee arthroplasties at Guys
Hospital in 1998, the measured blood loss ranged from
50 to 2,590 mL with an average of 780 mL. When calculated
(based upon pre- and postoperative Hb and the transfusion
given) this average was closer to 1,300 mL. The average loss
in males was much higher than in females 980 mL vs 650 mL.
Chapter 14  Transfusion Practice in Arthroplasty  49
Typically the loss and transfusion rate is less than total hip
arthroplasty but a 40–50% rate is not uncommon. In a study
of 477 patients only the preoperative Hb was a predictor of
transfusion risk. If > 13.5 g/dL there was only a 2.8% risk. This
actually could produce a lower complication rate but is not
generally accepted as good practise.9
Hemodilution and intraoperative salvage are not
appropriate for most primary TKAs as many are performed
under tourniquet and the blood loss and transfusion rate is
not particularly high in most cases.
Bilateral Total Knee Arthroplasty
The blood loss is significantly higher than that seen in
unilateral cases leading to an increased transfusion rate. In
patients with no predonation this rises from 18 to 57%. This
necessitates the application of one or more techniques to
avoid allogenic blood. There was a low rate of complications
and good clinical outcome in all groups at a mean follow-
up of four years (1 to 7.2). The group staged at a one-week
interval had the least blood loss (p = 0.004).10
In a comparative study of tranexamic acid (TEA) against
control showed significant reduction in blood loss. The total
postoperative drain output was found to be lower in patients
who received TEA as compared to the control group (275 mL
vs 810 mL) and this relation was also found to be statistically
significant (p = 0.000). The sample size was large enough for
this conclusion to be made (power 1.00 in all). This implied a
decrease in total blood loss in patients who were administered
TEA during bilateral total knee arthroplasty (TKA). As such
the amount of allogenic blood transfusion (BT) requirements
were also reduced in these patients as compared to the
control group (0.80 units vs 3.17 units).11
Patients with osteoarthritis of the knees were injected
with 5 mL (25 mg/mL) TEA and 5 mL analgesic containing
epinephrine (3 μg/mL) solution were injected at several
points into the posterior capsule before installation of the
prosthesis. The femoral medullar canal was closed with
autograft bone and then sealed compressively with cement.
Before the tourniquet was released, 10 mL TEA solution and
10 mL analgesic containing epinephrine were injected at
several points into the periosteum, synovium, joint capsule,
tendons, and deep fascia tissue (injection of analgesic
containing epinephrine into subcutaneous fat and dermis
was avoided). The residual nail holes in the bone and the
uncovered bone section were covered with bone wax. TEA
solution (20 mL) was injected into the articular cavity after
wound closure. The drainage tube was clamped for 4 hours,
then opened. Intraoperative blood loss, drainage volume, total
postoperative blood loss, and number of patients requiring
allogenic blood transfusion were significantly reduced.12 Cell
saver techniques for blood salvage is an important component
is reducing blood loss. OrthoPAT system is an important tool
in autoblood transfusion intraoperatively.13
50  Section 1  Clinical Hemotherapy
REFERENCES
1. Bierbaum MD, et al. An Analysis of Blood Management in
Patients Having a Total Hip or Knee Arthroplasty. J Bone Joint
Surg. 1999;81a:2-10.
2. de Andrade JR, Jove M, Landon G, Frei D, Guilfoyle M, Young
DC. Baseline hamoglobin as a predictor of risk of transfusion
and response to epoetin alfa in orthopedic surgery patient. Am
J Orthop. 1996;25:533-42.
3. Faris PM, Ritter MA, Abels RI. The American Erythropoetin
Study Group. The effects of recombinant human erythropoietin
on perioperative transfusion requirements in patients having
a major orthopedic operation. J Bone Joint Surg. 1996;78-A:
62-72.
4. Nuttall GA, Santrach PJ, Oliver WC Jr, Horlocker TT, Shaugh­
nessy WJ, Cabanela ME, et al. The predictors of red cell
transfusions in total hip arthroplasties. Transfusion. 1996;36:
144-9.
5. Walker RW, et al. Blood loss during primary total hip arthro­
plasty: use of preoperative measurements to predict the need
for transfusion. Ann R Coll Surg Engl. 1997;79:438-40.
6. Sharrock NE, Minoe R, Urquhart B, Salvati EA. The effect of two
levels of hypotension on intraoperative blood loss during total
hip arthroplasty performed under lumbar epidural anesthesia.
Anesthesia Analgesia. 1993;76:580-4.
7. Tric ME, Walker RH, DLima DD Morris BA, Colwell CW,
Jr. Blood Loss and Transfusion Rate in Noncemented and
Cemented/Hybrid Total Hip Arthoplasty. Is there a difference?
A comparison of 25 matched pairs. Orthopedics. 1999;22:S141.
8. Chandrataya A, et al. To drain or not drain: literature versus
practice. JR Coll Surg Edinb. 1998;43:404-6.
9. Lotke, Garino JP, et al. Autologous blood after total knee
arthoplasty: Reduction in non-surgical complications.
Presented at Am Acad Orthop Surg. 1997.p.265.
10. Forster MC, Bauze AJ, Bailie AG, Falworth MS, Oakeshott
RD. A retrospective comparative study of bilateral total knee
replacement staged at a one-week interval. JBJS (Br) 2006;88-
B(8):1006-10.
11. Dhillon MS, Bali K, Prabhakar S. Tranexamic acid for control of
blood loss in bilateral total knee replacement in a single stage.
Indian Journal of Orthopedics. 2011;45(2):148-52.
12. Zhaohui Liu, Wanshou Guo, Qidong Zhang, Guangduo Zhu.
Topical hemostatic procedures control blood loss in
bilateral cemented single-stage total knee arthroplasty.
Journal of Orthopedic Science. 2014.
13. Kyle C, Sinclair BS, Henry D, Clarke MD, Brie N, Noble BS.
Blood management in total knee arthroplasty: a comparison of
techniques. Orthopedics. 2009;32(1):19.
Restricting Transfusion
Requirements in Major Orthopedic
Surgery and its Alternatives
Ravisha Bhardwaj, RS Boparai
Introduction
In major orthopedic surgical procedures, perioperative
blood loss and blunted postoperative erythropoiesis, due to
surgery-induced inflammation, may lead to postoperative
anemia in almost 90% of patients. Allogenic blood transfusion
is the most frequently used measure for treating acute intra-
and postoperative anemia, especially in patients with low
preoperative hemoglobin levels and/or undergoing non-
elective surgery, as it may increase the patients’ hemoglobin
levels though transitorily, quickly and effectively.1
The rationale behind allogenic blood transfusion is to
restore oxygen delivery and provide a reserve, should further
bleeding occur. It is generally assumed that, in the event of
tissue hypoxia, the benefits on survival conferred by the
allogenic transfusion in certain patients clearly outweigh
the risks. This goal can only be achieved if dual indicators for
blood transfusion (level of oxygen carriers and evidence of
the oxygen tissue debt of anemic origin) are used. However,
in everyday clinical practice, reliable indicators of oxygen
deficit of anemic origin (mixed venous blood saturation,
partial tissue oxygen pressure, etc.) are often not available2
and, consequently, decisions on transfusion are usually
made just on the basis of the patient’s hemoglobin level
and/or symptoms. Thus, many allogenic blood transfusions
are unnecessarily given to patients with only relatively low
hemoglobin levels, just expecting that transfusion of red
blood cells will increase oxygen transport, thus alleviating
tissue hypoxia and improving outcome. Evidence based
medicine shows that allogenic blood transfusion produces a
variable increment in brain tissue oxygenation, as assessed
by direct measurement of partial tissue oxygen pressure, in
patients with neurological trauma and documented oxygen
deficit.3
As a result of improved screening for detecting trans-
fusion transmitted infections and better product conserva-
tion methods, allogenic blood transfusion is considered a
safe treatment option in developed countries. However, as
15
a biological product, allogenic blood transfusion will never
be risk-free: (i) screening methods for the detection of new
infectious threats will only be implemented once a significant
proportion of the transfused population has already been
infected; (ii) “clerical mistakes” and administration of
“wrong blood” are still too frequent (1/15,000–1/20,000); and
(iii) the risk of complications such as graft-versus-host
disease, metabolic disorders, bacterial contamination,
transfusion-related acute lung injury (TRALI), transfusion-
associated circulatory overload (TACO), and transfusion-
related immunomodulation (TRIM) still persists.4
In order to reduce variability in transfusion practice,
both in the proportions of patients receiving allogenic
blood transfusion and in the volume of blood administered
per transfused patient, scientific societies have developed
evidence-based guidelines and recommendations on the
indications of allogenic blood transfusion. The final objective
of these guidelines is a more rational and “restrictive” use of
allogenic blood transfusion in patients for whom pharma­
cological options are not available or cannot be implemented
(e.g. acute severe anemia).1 Accordingly, in making a
transfusion decision in euvolemic, nonbleeding patients:
• The risk of anemia and the risks and benefits of red
cell transfusion should be carefully balanced for each
individual patient;
• The so-called “liberal” transfusion protocols (pre-
transfusion hemoglobin concentration >9–10 g/dL)
should be generally avoided;
• Should allogenic blood transfusion be deemed necessary,
single unit transfusions are desirable; and
• Patients should be reassessed between transfusions to
determine the remaining transfusion needs.
In this regard, the transfusion requirements in critical
care (TRICC) trial demonstrated that the introduction of a
restrictive  transfusion protocol with transfusion trigger of
Hb <7 g/dL, reduced the rate of allogenic blood transfusions
by 33% and the allogenic blood transfusion index by 3 units
per patient compared to a liberal transfusion protocol with
52  Section 1  Clinical Hemotherapy
transfusion trigger of Hb <10 g/dL. There were no differences
in mortality rates between groups, not even for the subgroup
of patients with significant cardiac disease, but the restrictive
protocol resulted in a lower mortality rate among patients
who were younger (<55 years) or less critically ill (APACHE
score <20).5
However, blood transfusion conceals a number of well-
recognized risks and complications and blood products have
become more expensive because of their specific preparation
procedure. Even small changes to routine procedures can
lead to enormous benefits for patients, physicians, hospitals
and society in general.
Orthopedic surgery can be divided into the fields of elective
orthopedics and trauma. The elective aspects, particularly
joint replacement and spinal surgery, are well suited to the
many techniques available to help reduce the use of allogenic
blood. Trauma by definition is acute, unpredictable and a
much bigger challenge.
Techniques aPPLICAble to
orthopedic surgery6
In USA and many European countries predonation of blood
for autologous transfusion is perhaps the most widely utilized
method. Lowering the transfusion trigger is the simplest way
to reduce allogenic transfusion. Too often blood is given to
patients on an empirical basis. At times patients undergoing
total knee arthroplasty with a tourniquet inflated were
transfused even before any blood was lost. It is essential to
educate the relevant staff members who may order this trans­
fusion. This may be the anesthetist, the junior surgical staff
or, not infrequently, the surgeon himself who is unwilling
to change his practice despite overwhelming evidence to
the contrary. Secondly we can make the choice of surgical
procedure, which reduces blood loss like the use of closed
techniques for nailing long bone fractures. Similarly, the
use of external fixators for unstable pelvic fractures not only
reduces blood loss but at times can be life saving. Thirdly,
preoperative planning and rehearsal like surgeon being
familiar with the patient, the procedure, the instruments and
the implants can save valuable time and reduce blood loss.
Modification of surgical techniques is other very simple and
cost-effective way of reducing the use of allogenic blood.
Adherence to good surgical principles like taking more time
to identify and control bleeding vessels before the surgery
proceeds. Many procedures have their own potential
problems. Perforating branches of profunda femoris artery
are often injured when approaching the shaft of the femur.
Branches of the circumflex vessels are at risk with a posterior
approach to the hip. The superior lateral geniculate artery
may be cut during knee surgery. A few extra seconds to locate
and avoid or control these arteries can make a big difference
to overall blood loss. Appropriate positioning particularly
helpful in spinal surgery where the intra-abdominal pressure
can be lowered with the use of special frames or supports thus
preventing congestion of the epidural veins.
Choice of Anesthesia
It has long been recognized that spinal anesthesia for total hip
replacement surgery can have significant advantages. More
recently the beneficial effect of hypotension on intraoperative
blood loss during total hip surgery under epidural anesthesia
has been reported. The surgical blood loss was reduced from
700 mL to approximately 250 mL.7
Perioperative Use of Pharmacological Agents
There is only limited experience in the use of these agents
in orthopedic surgery. It is suggested that the use of
Desmopressin with Harrington rod spinal fusion surgery can
reduce blood loss by 32.5%. No benefit was noted with total
hip surgery however. Aprotinin has been reported to reduce
blood loss in total hip arthroplasty.
Perioperative Erythropoietin
Many studies have confirmed the safety and efficiency
of recombinant human erythropoietin. Used alone in
the perioperative period or as an adjunct to maximize a
predonation program, red blood cell production is increased
and allogenic transfusions are reduced.
Preoperative Optimization of Medical Status
The majority of patients undergoing orthopedic surgery
have a number of comorbid conditions, which need to be
addressed preoperatively. Probably half are hypertensive.
Many are taking aspirin, nonsteroidal anti-inflammatory
agents or anticoagulants. Coagulopathies should be recog­
ni­zed and treated.
Predonation of Autologous Blood
It has been the standard of care in many orthopedic units
across the world for a number of years. Although generally
effective there are increasing concerns related to cost factors,
the increase in preoperative anemia and the large number of
wasted units.
Hemodilution Techniques
As yet there is only limited information available in the
orthopedic setting. There are probably indications for its use
when blood loss is expected to be moderately high and the
patient has no coronary artery, renal, hepatic or pulmonary
disease.
 Chapter 15  Restricting Transfusion Requirements in Major Orthopedic Surgery and its Alternatives  53
Blood Salvage Intra- and Postoperatively
Simple drainage systems which collect, filter and reinfuse
shed blood postoperatively are very popular. It is possible
to reinfuse an average of 437 mL after primary total hip
replacement and 883 mL after primary total knee replace­
ment.8 Intraoperative salvage is much more costly in the
use of time and staff and has more limited applications. It is
perhaps useful for extensive spinal surgery.
Orthopedic Trauma
This is a particularly difficult area for effective blood
management. Many patients are elderly and frail with
multiple comorbidities and frequently anemic at the time
of injury hence needing allogenic blood transfusion. Pelvic
fractures are associated with major blood loss with transfusion
requirements ranging from 3.6 units to 14,8 units (average
5.9 units). Acetabular fracture fixation is associated with
transfusion in over 50% of cases. After initial stabilization,
early fixation of long bone fractures will reduce the blood
loss as well as reducing other complications viz lung injury.
After closed techniques and the use of fixators will reduce
the operative blood loss. Further surgery is often required
during the first few days with further blood loss. Later, the
problems of rehabilitation arise. Much work is now being
done on the effects of anemia on postoperative recovery,
vigour, length of hospital stay and costs.9 It is increasingly
clear that allogenic transfusion can have detrimental effects
on the overall condition of the patient. The infection rate is
probably doubled; not just the orthopedic wound but also
the rate of chest and urinary tract infection. Unfortunately
the nature and timing of these injuries precludes the use of
many modalities. Predonation is obviously impossible and
hemodilution and hypotensive techniques can be risky in
these patients.
Allogenic blood is still necessary in a number of cases
despite the potential drawbacks. This may be given in the
acute phase, i.e. pre or intraoperatively or later to correct the
problems associated with severe anemia.
Blood salvage can be of value in cases of high blood loss
such as pelvic fracture fixation. Most blood loss will occur
intraoperatively but postoperative salvage drains can be
of additional help with up to half the loss recoverable.
Erythropoietin may have great potential in orthopedic trauma
but studies are still limited. Early studies suggest a reduced
transfusion rate when given as 5 daily doses to hip fracture
patients undergoing operative fixation.10 In more major
cases it could only be used in the postoperative period but
should also help reduce the transfusion rate by improving the
erythropoietic recovery and help to avoid the complications,
particularly the increased infection rate.
FUTURE
It has taken many years for orthopedic surgeons to realize
the importance of an effective blood management program.
Even now many centers have minimal planning and rely
heavily on allogenic blood. It has been clearly shown
that even the simplest techniques such as lowering the
transfusion trigger can, at times, halve the transfusion rate.
In the immediate future, much can be achieved with good
education making surgeons better aware of the dangers of
allogenic transfusion and the options already available to
reduce its use. Predonation programs will likely continue
in the foreseeable future but much can be done to refine
them. Erythropoietin in the perioperative period has many
potential benefits. Blood substitutes have gone through a
long and difficult testing and validation process and early
clinical testing is underway. The process of disseminating
this knowledge should become simpler. The Internet and
electronic publishing will provide an easily accessible source
of information, which can be rapidly updated. Laptop or
hand-held computer based algorithms are being developed
for use in general clinical practice and also as research
tools.11
Conclusion
It will be some time before the concept of bloodless surgery
is finally realized but great strides have already been made.
The initial problem of making the orthopedic community
aware of the risks of allogenic transfusion is being solved
with better education. A wide array of treatment options is
now available and the indications for and effects of each are
being defined more accurately. The future is increasingly
exciting, as blood substitutes become available. Until that
time, we must strive to make the best use of the techniques
already available in order to achieve the best possible
outcome for our patients.
References
1. Manuel Muñoz and Santiago Ramón Leal-Noval. Restrictive
transfusion triggers in major orthopedic surgery: effective and
safe? Blood Transfus. 2013;11(2):169-71. (http://www.ncbi.
nlm.nih.gov/pmc/articles/PMC3626465/)
2. Vallet B, Adamczyk S, Barreau O, et al. Physiologic trans­fusion
triggers. Best Pract Res Clin Anesthesiol. 2007;21:173-81.
[PubMed]
3. Leal-Noval SR, Muñoz-Gómez M, Arellano-Orden V, et al.
Impact of age of transfused blood on cerebral oxygenation
in male patients with severe traumatic brain injury. Crit Care
Med. 2008;38:1290-6. [PubMed]
4. Annual SHOT report 2011. [Accessed on 26/11/2012]. Available
at: http://www.shotuk.org/wp-content/uploads/2012/07/
54  Section 1  Clinical Hemotherapy
SHOT-ANNUAL REPORT_Final Web Version Bookmarked_
2012_06_22.pdf.
5. Hébert PC, Wells G, Blajchman MA, et al. A multicenter,
randomized, controlled clinical trial of transfusion require­
ments in critical care. N Engl J Med. 1999;340:409-17.
6. Transfusion Alternatives in Orthopedic Surgery | Nataonline
www.nataonline.com/np/445/transfusion-alternatives-
orthopedic-surgery.
7. Sculco T, Ranawat C. The use of spinal anesthesia for total hip
replacement. J Bone Joint Surg. 1975;57:173-7.
8. Sharrock NE, Minoe R, Urquhart B, Salvati EA. The effect of two
levels of hypotension on intraoperative blood loss during total
hip arthroplasty performed under lumbar epidural anesthesia.
Anesthesia Analgesia. 1993;76:580-4.
9. Keating EM, Ranawat CS, Cats-Baril, W. Assessment of
post­­­
operative vigor in patients undergoing elective total
joint arthroplasty: A concise patient and caregiver-based
instrument. Orthopedics. 1999; 22: S119.
10. Goodnough LT, Merkel K. Parenteral iron and recombinant
human erythropoietin therapy to stimulate erythropoiesis in
patients undergoing repair of hip fracture. Hematology. 1996;
1:163-6.
11. Han CD, Shin DE. Postoperative blood salvage and reinfusion
after total joint arthroplasty. J Arthroplasty. 1997;12:511-6.
Transfusion Support
in Neurosurgery
Sandeep Kundra
Introduction
Neurosurgical procedures involve surgery on head and neck
or spine. Brain is a highly vascular organ and is supplied by
internal carotid arteries and vertebral arteries which form an
anastomosis named, the ‘circle of Willis’ at the base of brain.
Overall, brain receives roughly 15–20% of cardiac output.
Brain is a metabolically highly active organ, with a high
requirement for oxygen.
The result of these things is two fold:
1. Any injury or surgery on brain leads to greater bleeding as
compared to other body organs.
2. Blood loss which results in decreased oxygen carrying
capacity of brain may cause irreversible brain injury.
Among the neurosurgical techniques, surgeries on
cranium accounts for greater blood loss as compared to
spine. Among the cranial surgeries, meningiomas being more
vascular, tend to bleed more.
The purpose of this presentation is to highlight some of
the recent work and issues surrounding the factors, which
must be considered when making a decision to transfuse
a neurosurgical patient. The focus is on the perioperative
period. As with all treatment decisions, transfusion decisions
must be taken in the context of the individual patient’s co-
morbidities and the clinical scenario, such as ongoing
bleeding. Although the issues discussed center primarily
on the use of blood components and derived products, the
contributions of other equally important factors such as
surgical hemostasis, temperature, pH, and serum calcium
must be acknowledged lest we neglect to consider them
during our clinical decision-making.
Threshold for transfusion in
Neurosurgery is uncertain
The threshold hemoglobin at which transfusion should be
performed in neurosurgical patients is very uncertain. In
healthy population, Hopf et al.1 demonstrated that acute
hemodilution down to 5 g/dL in healthy volunteers does
16
not compromise subcutaneous tissue oxygen tension due
to compensation with increasing blood flow. However,
Weiskopf, et al.2,3 showed that cognitive abilities are
compro­mised by acute hemodilution below 6 g/dL. Thus,
the application of end point for transfusion at Hb of 6 g/
dL, in the context of injured brain is fraught with risks. The
possibility of worsening brain injury has been demonstrated
even at hemoglobin levels above the commonly accepted
threshold of 7.0 g/dL.4 Decisions about when to transfuse
neurosurgical patients must further weigh the putative
benefits of increasing oxygen carrying capacity and delivery
against our increasing understanding of the serious risks
associated with the transfusion of blood products. These risks
include infection, hemolysis, transfusion-related acute lung
injury, alloimmunization, and immunosuppression,5 as well
as concerns about whether or not transfused red cell products
effectively augment oxygen delivery.6,7
Neurosurgery differs from other surgical specialties on
three main accounts:
1. Being highly vascular structures, surgery on nervous tissue
entails significant blood loss which needs to be replaced.
2. Patients can bleed to death in a very short time if accidental
injury to venous sinuses or arterial vasculature of brain
occurs.
3. Many patients need critical care, wherein, most critical
care specialists tend to keep the Hb around 10 g/dL.
So, most neurosurgeons and neuroanesthetists tend
to transfuse blood at much higher Hb levels of usually 8 or
9 g/dL.
The need for blood product transfusion in neurosurgery
can broadly be divided into three categories:
1. For acute blood loss.
2. For correcting chronic anemia prior to taking up for
surgery.
3. Correcting coagulation defects.
To highlight clinical relevance, information is presented
according to presentations and procedures common to
neuroanesthetic practice.
56  Section 1  Clinical Hemotherapy
Cerebrovascular Surgery
The incidence of intraoperative transfusion for cerebro­
vascular procedures such as aneurysm clipping and carotid
endarterectomy has been described as relatively low, with
some published reports of intraoperative transfusion rates
well under 10%.8 When the preoperative and postoperative
phases are taken into account, transfusion rates are higher.
Ruptured intracranial aneurysms represent a unique
circumstance as classically treatment involves treatment with
induced hypertension and hypervolemia and hemodilution.
The rationale for this treatment is to strike a balance between
reduced blood viscosity and preserved oxygen carrying
capacity. In spite of this theory, hemodilution as a strategy to
improve outcome in symptomatic vasospasm has been called
into question.9,10 There is evidence that higher hemoglobin
levels in patients with subarachnoid hemorrhage (SAH) is
associated with improved outcome.11 Although there seems
to be reasonable evidence to suggest that higher hemoglobin
levels are good, it is not clear what hemoglobin level is
optimal. A recent study has identified a cut-off of 9 g/dL, or
hematocrit of 0.27, below which an increased incidence of
cerebral hypoxia is observed via invasive monitors of brain
tissue oxygenation.12 To further complicate matters, it is
unclear as to whether or not it is useful to achieve or maintain
higher hemoglobin levels through transfusion. It has been
asserted that transfusion in the setting of SAH in particular,
may be associated with increased risk for vasospasm (at least
radiographically, if not clinically)13 and poor outcome. So
most neurointensivists tend to target a hemotocrit of 27–30%,
albeit the evidence to support or refute this practice is lacking.
Intracerebral Hemorrhage
Intracerebral hemorrhage (ICH) is most commonly due
to hypertension. Sometimes, patients on anticoagulant
therapy may also develop ICH. Surgery is performed when
ICH is causing mass effect resulting in increased intracranial
pressure (ICP). Transfusion depends on the amount of blood
loss and the pre-existing Hb with most neuroanesthetists
targeting a Hb of 9 g/dL. The most significant recent
development in the treatment of intracerebral hemorrhage
(ICH) has been the result of the phase III FAST trial,
investigating the role of activated factor VII in treatment.14
Although hematoma growth was reduced, there was no
demonstrated benefit for survival or functional outcome.
Traumatic Brain Injury
Traumatic brain injury (TBI) represents a complex and
heterogeneous group of patients in which mechanisms
that normally help to ensure an adequate supply of blood
and oxygen to brain parenchyma, such as autoregulation,
are variably impaired. It may seem logical that higher
hemoglobin levels and greater oxygen carrying capacity
would be desirable under this circumstance. Again, the exact
hemoglobin value below which one should be concerned is
uncertain. A subset analysis of head injured patients from the
transfusion requirements in critical care trial15 did not detect
a difference between populations maintained at hemoglobin
of 7.0–9.0 g/dL versus 10.0–12.0 g/dL,16 though it may have
been somewhat underpowered. A number of studies have
demonstrated an apparent improvement in cerebral well
being after transfusion based on available neurophysiologic
monitors17,18 whereas other studies looking at clinical
outcome have demonstrated a lack of improvement with
transfusion.19,20 Taken together, the appropriate course
of action becomes even less evident. Transfusion of
blood components has an additional role in TBI due to
the association of coagulopathy with brain injury, and its
association with poor outcome.21-23 Brain parenchyma is a
rich source of tissue factor, which activates coagulation when
released. By this mechanism, disseminated intravascular
coagulation (DIC) may be induced. It has been suggested
that this clotting activation may in some cases be matched by
fibrinolysis, resulting in a net consumption of clotting factors,
but possibly avoiding the microembolic phenomenon
of DIC. Owing to the complex, heterogeneous, and likely
multifactorial nature of the coagulopathy associated with
TBI, it is not surprising that guidelines for treatment of
coagulopathy caused by brain injury do not exist. Depending
on the mechanism contributing to the problem, blood
products such as plasma, platelets, and cryoprecipitate likely
have a role in replacement of depleted coagulation substrate.
The association of TBI with the multiply injured poly-
trauma patient also merits mention. Brain injury is often not
the only source of bleeding and coagulopathy, as a significant
proportion of brain injured patients may present with other
injuries. Recent literature regarding massive transfusion in
trauma has focused on the role of increased ratios of plasma
and platelets to red blood cells.24 In the context of closed head
injury, avoidance of coagulopathy or its rapid and adequate
reversal is arguably even more important.
Tumor Surgery
As with traumatic brain injury, resection of brain tumors
has been associated with a clinically significant incidence
of DIC.25,26 The development of coagulopathy in the context
of tumor resection is again associated with poor outcome.
One issue regarding transfusion, which is particular to tumor
surgery, is the role it may play in tumor progression.27 It is
speculated that the immune-modulatory effects of trans-
fusion impair the body’s innate abilities to suppress
tumor growth and spread. The exact significance of these
observations as applied to brain metastases or primary
neurological tumors remains to be established. For now, it
is at the minimum yet another reason why we must strive to
avoid unnecessary transfusion.

Interventional Neuroradiologic Procedures
(Embolization, Acute Stroke)
Advances in the field of interventional radiology continue to
have an important impact on procedures which pose a risk
for perioperative hemorrhage. Preoperative embolization of
tumors and arteriovenous malformations (AVMs) can greatly
attenuate blood loss at the time of surgery, and reduce the
requirement for transfusion. Advances in the field naturally
lead to more questions. What International Normalized
Ratio (INR) or platelet count is appropriate for a patient
undergoing embolization of an AVM or treatment for acute
stroke? Patients presenting with coagulopathy enrolled in
the MERCI and multi-MERCI trials underwent interventional
radiology procedures for cerebral artery revascularization
without pre-procedure correction of their coagulation status,
and did not demonstrate any higher rate of symptomatic
intracranial hemorrhage.28 A hemoglobin target that is most
appropriate for a patient undergoing an intervention for acute
embolic stroke is unknown. Most of the work to date suggests
that extremes of hematocrit are poor prognostic indicators
in acute stroke. The study by Allport et al.29 showed reduced
reperfusion and tissue survival with increasing hematocrit
in a patient population with median hematocrit levels of
39–42%. Hemodilution for the purpose of reduced viscosity
and improved flow through narrowed vessels has been shown
to lack efficacy as well.30
Anticoagulated Patient
Patients treated with vitamin K antagonists such as warfarin
need special care when they present for emergency neuro­
surgical procedures. Whether it be intracerebral hemorrhage,
trauma, or subarachnoid hemorrhage, these patients require
rapid reversal of their anticoagulation. Supplementation
with vitamin K, although appropriate, does not act as
quickly as one might like in these situations. Replenishment
of vitamin K dependent clotting factors with plasma has
long been viewed as appropriate in these patients, but
may not provide reversal in the time frame desired,31,32
and alternatives have been advocated.33,34 Prothrombin
complex concentrates (PCCs) provide more rapid reversal
of vitamin K antagonists, are quick to prepare, are virally
inactivated, and do not require the volume load of plasma.
There is, however, no evidence for improved outcomes
in patients treated with PCC versus plasma. The exact
components of different PCC products vary by manufacturer,
and different products may not be equally effective in
restoring coagulation,35 so the clinician must be familiar
with the specific product stocked at one’s own institution.
Activated factor VII has also been used to rapidly reverse
coagulopathy associated with vitamin K antagonists and
intra­cranial bleeding.36 The decision as to whether one
should choose activated factor VII or PCCs is not entirely
Chapter 16  Transfusion Support in Neurosurgery  57
clear, although there are animal data to suggest that PCCs
may be more effective.37
Blood conservation strategies
Given many of the conflicting advantages and disadvantages
of blood transfusion in the neurosurgical patient, perhaps
an aggressive implementation of perioperative blood
conservation strategies is the answer. If transfusion counter­
acts the benefit of increased hemoglobin levels in the peri­
operative setting, one of the main interventions with which
we are left is the scrupulous avoidance of activities which
contribute to anemia. The goal of any blood conservation
strategy is to reduce or eliminate the exposure to allogeneic
blood. Autologous predonation, erythropoietic support,
acute normovolemic hemodilution, intraoperative cell
salvage, induced hypotension, and the use of pharmacological
agents in addition to meticulous surgical hemostasis have
all been associated with reductions in allogeneic blood
use, yet the cost effectiveness of these strategies has been
questioned.38,39 Many of these techniques have been adopted
into use in neurosurgery; however, few have been studied in
this patient population. Limited evidence appears to show
that intraoperative cell salvage and perioperative autologous
transfusion is well-tolerated and offers a modest reduction
in allogeneic blood transfusion rates during intracranial
surgery.40 The antifibrinolytics epsilon-aminocaproic acid
and tranexamic acid have been studied in the setting of
preventing rebleeding of aneurysmal acute subarachnoid
hemorrhage. Both agents have shown considerable reduction
in rebleeding rates; however, high dose and prolonged use
(>72 hours) in this population is associated with increased
risk of vasospasm and hydrocephalus negating any overall
benefit. These conclusions were drawn in a time when early
surgical aneurysm clipping was not recommended. Both
epsilon aminocaproic acid and tranexamic acid have been
re-evaluated in view of changes in surgical management of
aneurysmal subarachnoid hemorrhage and have shown
positive results in reducing early rebleeding rates yet sparing
the complications found in earlier work.41,42
Conclusion
It is known that in the context of neurosurgery, higher
hemoglobin levels are associated with better outcomes in
general. Unfortunately, maintaining these levels through
transfusion of banked, allogeneic red blood cells appears to
be associated with worse outcome. As neuro-anesthetists,
we can contribute by minimizing unnecessary losses,
such as increased bleeding from venous congestion, and
avoiding superfluous blood draws, although the latter issue
is perhaps more applicable over the longer term in the ICU
environment. Notable questions for the future include
the exact role of transfusion sparing practices such as cell
58  Section 1  Clinical Hemotherapy
salvage or acute normovolemic hemodilution, and whether
or not they may be associated with improved neurological
outcomes as compared with allogeneic transfusion. The role
of strategies to address anemia preoperatively prior to elective
neurosurgical procedures also remains to be addressed with
regard to outcome.
A great deal of focus has been directed toward monitoring
modalities such as:
• Brain tissue oxygen tension
• Near infrared spectroscopy
• Jugular bulb catheter sampling, to name just a few.
Indications from these monitors of inadequate regional
or global oxygenation have been used to advocate for
transfusion, with a resultant improvement in the monitored
parameter. Increased use of these monitors may aid us in
balancing cerebral requirement of blood thus optimizing
transfusion. Once issues such as reduced perfusion pressure,
vasospasm, pyrexia, and seizures have been addressed
without a satisfactory improvement in monitored parameters,
one might reasonably consider transfusion.
As for the exact number to target, we feel that the avail-
able evidence currently supports a hemoglobin level of
8.0–9.0 g/dL. In the context of ongoing bleeding in the
operating room in particular, targeting a lower number
(such as 7.0 g/dL) may result in brief periods of even more
profound anemia, which appear to be clearly linked with
inadequate brain perfusion. It cannot be stressed strongly
enough, however, that these targets may need to be modified
or revised in the context of significant comorbidities such as
coronary artery disease or hypoxemia. We must continue to
examine these issues to identify which patient groups in the
heterogeneous neurosurgical population will benefit from
the interventions that we might offer.
References
1. Hopf HW, Viele M, Watson JJ, et al. Subcutaneous perfusion and
oxygen during acute severe isovolemic hemodilution in healthy
volunteers. Arch Surg (Chicago, Ill: 1960). 2000;135:1443–9.
2. Weiskopf RB, Kramer JH, Viele M, et al. Acute severe isovolemic
anemia impairs cognitive function and memory in humans.
Anesthesiology. 2000;92:1646-52.
3. Weiskopf RB, Feiner J, Hopf HW, et al. Oxygen reverses deficits
of cognitive function and memory and increased heart rate
induced by acute severe isovolemic anemia. Anesthesiology.
2002;96:871-7.
4. Hare GMT, Tsui AKY, McLaren AT, et al. Anemia and cerebral
outcomes: many questions, fewer answers. Anesth Analg.
2008;107:1356-70.
5. Hendrickson JE, Hillyer CD. Noninfectious serious hazards of
transfusion. Anesth Analg. 2009;108:759-69.
6. Weiskopf RB, Feiner J, Hopf H, et al. Fresh blood and aged
stored blood are equally efficacious in immediately reversing
anemia-induced brain oxygenation deficits in humans.
Anesthesiology. 2006;104:911-20.
7. Tinmouth A, Fergusson D, Yee IC, He´ bert PC. Clinical
consequences of red cell storage in the critically ill. Transfusion.
2006;46:2014-27.
8. Couture DE, Ellegala DB, Dumont AS, et al. Blood use in
cerebrovascular neurosurgery. Stroke. 2002;33:994-7.
9. Treggiari MM, Deem S. Which H is the most important in
triple-H therapy for cerebral vasospasm? Curr Opin Crit Care.
2009;15:83-6.
10. Ekelund A, Reinstrup P, Ryding E, et al. Effects of iso- and
hypervolemic hemodilution on regional cerebral blood
flow and oxygen delivery for patients with vasospasm after
aneurysmal subarachnoid hemorrhage. Acta Neurochir.
2002;144:703-12; discussion 712-713.
11. Naidech AM, Jovanovic B, Wartenberg KE, et al. Higher
hemoglobin is associated with improved outcome after
subarachnoid hemorrhage. Crit Care Med. 2007;35:2383-9.
12. Oddo M, Milby A, Chen I, et al. Hemoglobin concentration and
cerebral metabolism in patients with aneurysmal subarachnoid
hemorrhage. Stroke. 2009;40:1275-81.
13. Smith MJ, Le Roux PD, Elliott JP, et al. Blood transfusion
and increased risk for vasospasm and poor outcome after
subarachnoid hemorrhage. J Neurosurg. 2004;101:1-7.
14. Mayer SA, Brun NC, Begtrup K, et al. Efficacy and safety
of recombinant activated factor VII for acute intracerebral
hemorrhage. N Engl J Med. 2008;358:2127-37.
15. He´ bert PC, Wells G, Blajchman MA, et al. A multicenter,
randomized, controlled clinical trial of transfusion
requirements in critical care. Transfusion Requirements
in Critical Care Investigators, Canadian Critical Care Trials
Group. N Engl J Med. 1999;340:409-17.
16. McIntyre LA, Fergusson DA, Hutchison JS, et al. Effect of a
liberal versus restrictive transfusion strategy on mortality in
patients with moderate to severe head injury. Neurocrit Care.
2006;5:4-9.
17. Leal-Noval SR, Rinco´ n-Ferrari MD, Marin-Niebla A, et al.
ransfusion of erythrocyte concentrates produces a variable
increment on cerebral oxygenation in patients with severe
traumatic brain injury: a preliminary study. Intensive Care
Med. 2006;32:1733-40.
18. Zygun DA, Nortje J, Hutchinson PJ, et al. The effect of red blood
cell transfusion on cerebral oxygenation and metabolism after
severe traumatic brain injury. Crit Care Med. 2009;37:1074-8.
19. George ME, Skarda DE, Watts CR, et al. Aggressive red blood
cell transfusion: no association with improved outcomes for
victims of isolated traumatic brain injury. Neurocrit Care.
2008;8:337-43.
20. Salim A, Hadjizacharia P, DuBose J, et al. Role of anemia in
traumatic brain injury. J Am Coll Surg. 2008;207:398-406.
21. Saggar V, Mittal RS. Hemostatic abnormalities in patients with
closed head injuries and their role in predicting early mortality.
J Neurotrauma. 2009;51(3):567-71.
22. Talving P, Benfield R, Hadjizacharia P, et al. Coagulopathy in
severe traumatic brain injury: a prospective study. J Trauma.
2009;66:55-61.
23. Pasternak JJ, Hertzfeldt DN, Stanger SR, et al. Disseminated
intravascular coagulation after craniotomy. J Neurosurg
Anesthesiol. 2008;20:15-20.
24. Holcomb JB, Wade CE, Michalek JE, et al. Increased plasma
and platelet to red blood cell ratios improves outcome in
466 massively transfused civilian trauma patients. Ann Surg.
2008;248:447-58.
25. Brecknell JE, McLean CA, Hirano H, et al. Dissemi-nated
intravascular coagulation complicating resection of a
malignant meningioma. Br J Neurosurg. 2006;20:239-41.

26. Eom KS, Kim JM, Kim TY. Disseminated intravascular coagula­
tion in a patient undergoing removal of metastatic brain tumor.
J Korean Neurosurg Soc. 2008;44:341-4.
27. Atzil S, Arad M, Glasner A, et al. Blood transfusion promotes
cancer progression: a critical role for aged erythrocytes.
Anesthesiology. 2008;109:989-97.
28. Nogueira RG, Smith WS. Safety and efficacy of endovascular
thrombectomy in patients with abnormal hemostasis:
pooled analysis of the MERCI and multi MERCI trials. Stroke.
2009;40:516-22.
29. Allport LE, Parsons MW, Butcher KS, et al. Elevated hematocrit
is associated with reduced reperfusion and tissue survival in
acute stroke. Neurology. 2005;65:1382-7.
30. Asplund K. Hemodilution for acute ischemic stroke. Cochrane
Database of Systematic Reviews 2002, Issue 4. Art. No.:
CD000103.
31. Aiyagari V, Testai FD. Correction of coagulopathy in warfarin
associated cerebral hemorrhage. Curr Opin Crit Care.
2009;15:87-92.
32. Lee SB, Manno EM, Layton KF, et al. Progression of warfarin
associated intracerebral hemorrhage after INR normalization
with FFP. Neurology. 2006;67:1272-4.
33. Ansell J, Hirsh J, Hylek E, et al. Pharmacology and management
of the vitamin K antagonists: American College of Chest
Physicians Evidence-Based Clinical Practice Guidelines (8th
Edition). Chest. 2008;133:160S-98S.
34. Levy JH, Tanaka KA, Dietrich W. Perioperative hemostatic
management of patients treated with vitamin K antagonists.
Anesthesiology. 2008; 109:918-926.
Chapter 16  Transfusion Support in Neurosurgery  59
35. Holland L, Warkentin TE, Refaai M, et al. Suboptimal effect of
a three-factor prothrombin complex concentrate (Profilnine-
SD) in correcting supratherapeutic international normalized
ratio due to warfarin overdose. Transfusion. 2009;49:1171-7.
36. Ilyas C, Beyer GM, Dutton RP, et al. Recombinant factor VIIa
for warfarin associated intracranial bleeding. J Clin Anesth.
2008;20:276-9.
37. Dickneite G. Prothrombin complex concentrate versus
recombinant factor VIIa for reversal of coumarin anticoagula­
tion. Thromb Res. 2007;119:643-51.
38. Goodnough LT, Brecher ME, Kanter MH, et al. Transfusion
medicine. Second of two parts: blood conservation. N Engl J
Med. 1999;340:525-33.
39. Goodnough LT, Monk TG. Blood conservation in patients
undergoing noncardiac surgery. Curr Opin Anaesthesiol.
2000;13:365-70.
40. Cataldi S, Bruder N, Dufour H, et al. Intraoperative autologous
blood transfusion in intracranial surgery. Neurosurgery.
1997;40:765-71.
41. Chwajol M, Starke RM, Kim GH, et al. Antifibrinolytic therapy
to prevent early rebleeding after subarachnoid hemorrhage.
Neurocrit Care. 2008;8:418-26.
42. Komotar RJ, Zacharia BE, Mocco J, et al. Controversies in the
surgical treatment of ruptured intracranial aneurysms: the
First Annual J. Lawrence Pool Memorial Research Symposium:
controversies in the management of cerebral aneurysms.
Neurosurgery. 2008;62:396-407.
 Maternal, Fetal and Neonatal Transfusion Practice
Noninvasive Prenatal Screening
for Rh Hemolytic Disease
Manjit Kaur Mohi
There is a fetal diagnostic technique available today that
involves analysing blood taken from the pregnant woman for
the genetic characteristics of her fetus. Noninvasive prenatal
diagnosis (NIPD), exploits the presence of cell-free fetal DNA
in the blood of the pregnant woman, and often eliminates the
need for invasive sampling of e.g. the placenta or amniotic
fluid. Small amounts of cell-free fetal DNA are present in
the pregnant woman’s plasma during pregnancy1 but are
rapidly eliminated from the mother’s circulation following
delivery.2 Fetal genetic testing and aneuploidy diagnosis
have until recently both needed invasive diagnostic sampling
procedures carrying a small but significant risk of miscarriage.
In 1997, the presence of cell-free fetal DNA (cffDNA) in the
maternal circulation was reported. Fetal RhD typing using
cffDNA to determine fetal blood group status from maternal
blood has been used since 2000 to direct the management
of pregnancies in RhD-negative women sensitized against
RhD,which are therefore at risk of hemolytic disease of the
fetus and newborn (HDFN).3
Fetal DNA comes from the placenta, can be detected from
the first trimester of pregnancy . Maternal blood is therefore a
reliable source of material for prenatal diagnosis.
However, the cffDNA is mixed with a larger proportion
of maternal cell-free DNA and current methodologies do
not allow complete separation of fetal from maternal DNA
in vitro. Therefore, the first applications of this phenomenon
focused on the detection, or exclusion of, paternally-inherited
fetal DNA sequences that are not present in the mother,
such as Y chromosome sequences in pregnancies with a
male fetus or rhesus D (RhD) sequences in women who are
RhD-negative. Recently, DNA sequencing technologies have
allowed very precise relative quantification of DNA fragments.
Noninvasive prenatal diagnosis (NIPD) was introduced a
few years ago in Sweden for fetal blood group determination
in pregnant women immunized against RhD or Rhc.
Determination of fetal blood group establishes whether or
not the fetus is at risk. If the fetus has a blood group antigen
against which the mother has antibodies, the woman needs
to be monitored with regular antenatal checks and, possibly,
17
treatment. If the fetus does not have any of these antigens,
further tests can be avoided and the parental anxiety in this
regard can be alleviated for the remainder of the pregnancy.
The use of NIPD can, therefore, help to improve the safety
of medical care offered to these women and their unborn
children.
In the UK, cffDNA testing has replaced amniocentesis for
fetal blood grouping. This is important because if the fetus
is RhD positive, amniocentesis for blood grouping risks
boosting the antibody titer and so converting mild disease
to severe. Determination of other fetal blood groups such as
Kell, C, c and E using cffDNA has also been reported.
The International Blood Group Reference Laboratory
in Bristol reported the successful development of a high-
throughput methodology using automated robotic techniques
suitable for mass screening of all women who are RhD-
negative. The use of cffDNA for fetal blood grouping has
therefore moved from use in women with the antibodies,
to routine antenatal care of RhD-negative women allowing
antenatal administration of anti-D immunoglobulin (a blood
product pooled from multipledonors) to be avoided in the
30–40% of RhD-negative women who are carrying RhD-
negative babies.4
NIPD can also be used to screen all RhD-negative pregnant
women,5 enabling the early determination of fetal blood
group. About 85% of the Swedish population is RhD-positive.
The other 15% of the population is RhD-negative and lacks
the RhD protein. About 60% of these RhD-negative women
will carry an RhD-positive baby. In Sweden, RhD prophylaxis
is given to these women after delivery, in order to reduce the
risk of RhD immunization.
If NIPD is to be offered in the future to all RhD-negative
pregnant women, the objective would be to give RhD
prophylaxis during pregnancy (antenatal prophylaxis) only
to those carrying an RhD-positive child, in order to reduce
the risk of RhD immunisation. The use of NIPD to screen
these women would, when combined with targeted RhD
prophylaxis, have the potential to reduce the number of new
cases of RhD-immunized women.6
 Chapter 17  Noninvasive Prenatal Screening for Rh Hemolytic Disease  61
Possible sources of error
Early gestational age, maternal obesity, multiple pregnancies
placental mosaicism, maternal conditions.
Ethical aspects
The high degree of accuracy of NIPT as early as 7 weeks of
pregnancy carries a number of socioethical implications,
such as the selective termination of fetuses according to sex.
Other issues subsequent to the removal of miscarriage risk
include the possibility that women may seek prenatal diagnosis
for an increasing number of conditions or for paternity testing.
Cost/availability
At present it seems that laboratories offering these services
are in limited geographical areas. In Europe, one of the first
laboratories was established in Germany.
Considering the number of tests that may now be request­
ed and the potential difficulties of transporting human blood
samples.
Conclusion
Blood Group Determination
Pregnant women immunized against RhD or other blood
group antigens RhD-negative pregnant women who are not
RhD immunized, i.e. screening.
Sex Determination
Pregnant women with a medical indication for fetal sex
determination as in, e.g. X-linked diseases.
References
1. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman
CW, et al. Presence of fetal DNA in maternal plasma and serum.
Lancet. 1997;350:485-7.
2. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM.
Rapid clearance of fetal DNA from maternal plasma. Am J Hum
Genet. 1999;64:218-24.
3. Illanes S, Soothill P. Management of red cell alloimmunisation
in pregnancy: the non-invasive monitoring of the disease.
Prenat Diagn. 2010;30:668-73.
4. Finning K, Martin P, Summers J, Massey E, Poole G, Daniels
G. Effect of high throughput RHD typing of fetal DNA in
maternal plasma on use of anti-RhD immunoglobulin in RhD
negative pregnant women: prospective feasibility study. BMJ.
2008;336:816-8.
5. Clausen FB, Christiansen M, Steffensen R, Jørgensen S,
Nielsen C, Jakobsen MA, et al. Report of the first nationally
implemented clinical routine screening for fetal RHD in D–
pregnant women to ascertain the requirement for antenatal
RhD prophylaxis. Transfusion. 2012;52:752-8.
6. de Haas M, van der Ploeg CP, Scheffer PG, Verlinden DA,
Hirschberg H, Abbink F, et al. A nation-wide fetal RHD
screening programme for targeted antenatal and postnatal
anti-D. ISBT Sci Ser. 2012;7:164-7.
Antenatal Antibody Screening
and Intrauterine Transfusion
in Pregnancy
Amit Aggarwal
Hemolytic disease of the fetus and newborn (HDFN) is a
condition in which passage of maternal antibodies through
placenta results in immune hemolysis of fetal/neonatal
red cells. HDFN is a challenge that continues to stalk
obstetricians and transfusion medicine specialists, even after
4 decades of introduction of RhD prophylaxis. Screening of all
antenatal women for red cell alloantibodies is mandatory in
developed countries. While guidelines for antibody screening
in pregnancy have been laid down by drug controller general,
India, they are not universally followed.
Years ago, HDFN was almost synonymous with RhD
alloimmunization and was a common neonatal problem.
The introduction of postnatal immunoprophylaxis in 1970
reduced the incidence of maternal D alloimmunization from
14% to 1–2%. Subsequently, antenatal immunoprophylaxis
was also started which further reduced Rh D alloimmunization
to 0.1%. In the western world, ABO incompatibility is now the
single largest cause of HDFN. However, in many developing
nations, anti D is still one of the common antibodies found in
pregnant women. Besides the anti D alloantibody, moderate-
severe HDFN cases attributed to other alloantibodies have
been described from Asian countries in the last decade. In
addition, new treatment modalities particularly improved
phototherapy and use of intravenous immunoglobulin (IVIG)
have altered the clinical course of the disease.
With routine immunoprophylaxis with Rh IG, ABO
incompatibility is now the single largest cause of HDFN in
the western world. ABO hemolytic disease occurs almost
exclusively in infants of blood group A or B born to O group
mothers, because Ig G anti A, anti B, occur more commonly in
group O than group A or B individuals. However, rare cases of
ABO incompatibility have been reported in group A2 mothers
with high titer anti B.
Antenatal testing and HDN status
in Developing countries
Many developed nations have national antenatal screening
programs such as, the Dutch screening program in
18
Netherlands and a similar screening program in Sweden.
However, in developing countries, anti D continues to be a
common alloantibody found in pregnant women. Failure
to administer Rh IG or antenatal sensitization prior to its
administration is the likely cause. Inadequate prenatal
care due to unawareness and financial constraints further
compound the problem. Besides anti D antibody, ABO
incompatibility and other alloantibodies have been reported
to cause severe HDFN in many Asian countries.
Reports from Sri Lanka, Bangladesh and India have
described unusually severe ABO-HDN requiring multiple
exchange transfusions. In a study from Saudi Arabia,
nearly one third of infants with ABO incompatibility
required exchange transfusions and the use of intravenous
immunoglobulin (IVIG) in such cases reduced the need
for exchange transfusion. Similarly IVIG has also been
used in Iran in Rh D HDN to reduce the need for exchange
transfusion. Asian and Black infants are known to have a
higher prevalence of DAT positive test than Caucasians. An
unusual report also describes severe ABO incompatibility in a
Bangladeshi B positive mother with an A1B infant.
Alloimmunization rates amongst pregnant women range
from 0.4 to 2.7% globally. The implicated antibodies could
be naturally occurring (anti A, anti B) or immune antibodies
which develop following a sensitizing event like transfusion
or pregnancy. The hemolytic process may result in anemia or
hyperbilirubinemia or both; thereby affecting fetal/neonatal
morbidity and mortality.
Many developed nations have implemented regular
screening of all pregnant women and even have national
screening programs. This is necessary to ensure timely
availability of antigen negative blood and reduce effects on
the newborn. Antenatal services in India are non-uniform
and do not have universal reach. We need to focus on
universal implementation of antibody screening and anti
D prophylaxis. Early detection and timely intervention can
help reduce morbidity and mortality (Table 1). Transfusion
services play a vital role in the antenatal detection, monitoring
and providing transfusion support to such cases.
 Chapter 18  Antenatal Antibody Screening and Intrauterine Transfusion in Pregnancy  63

Table 1: Recommended prenatal testing

Testing and condition Timing
Blood group (ABO and RhD)
All pregnancies Initial visit
Other For pretransfusion testing
Antibody screening
All pregnancies Intial visit
RhD negative females For those RhD negative females who will receive RhD-lg (at 28 weeks or at the time of any
sensitising event), the blood sample must be collected prior to its administration
Other For pretransfusion testing
Antibody identification Upon initial detection
Antibody titration/quantitation Rh antibodies and other potentially significant antibodies capable of causing HDN
In the event that clinically significant antibodies are detected, further testing is indicated at
intervals no greater than 4 weeks. Seek specialist advice.
Abbreviation: HDN­—Hemolytic disease of the newborn

Intrauterine Transfusion
Intrauterine transfusion (IUT) is a process where blood is
transfused to the fetus, in order to provide a better chance
of survival from life threatening conditions associated with
severe anemia, including Rh isoimmunization. The incidence
of Rh negativity in India is about 1–5% and the rate of Rh
sensitization to be approximately 0.79% of live births.
Intrauterine infusion of red blood cells (RBCs) into
the fetus is one of the most successful means of in utero
therapy. This has contributed to survival of several severely
anemic fetuses. Although universal use of Rh (D) immune
globulin has dramatically reduced the need for intrauterine
transfusions, the procedure continues to be an essential
modality for treatment of severe fetal anemia from a variety
of causes. IUT is indicated for fetal anemia resulting from
varied causes like blood group isoimmunization, parvovirus
B19 infection, fetomaternal hemorrhage, twin anemia-
polycythemia syndrome in complicated monochorionic twin
pregnancies, hemoglobinopathies, dyserythropoiesis, red-
cell enzymopathy like G6PD deficiency.
Intrauterine transfusion of RBCs is indicated to prevent
fetal death from severe anemia. The procedure is generally
limited to fetuses between 18 and 34 weeks of gestation
because of technical limitations before 18 weeks and
excessive fetal risk compared with delivery after 34 weeks. The
introduction of measures to both predict the severity of fetal
disease and treat it has greatly reduced perinatal mortality
rates and augmented survival rates.
Should titers rise to 1:16 or higher, fetal assessment is
indicated via noninvasive methods. The only definitive
means of diagnosing fetal anemia and acidosis is via fetal
blood sampling (FBS), also known as cordocentesis or
percutaneous umbilical blood sampling (PUBS),
The blood for possible transfusion must be typed and cross-
matched against the mother’s serum to help rule out any other
possible hemolytic antibodies. Additionally, the unit of red
blood cells should be cytomegalovirus-negative, irradiated,
less than 5 days old, and with a hematocrit level between 75
and 80%. This can require several hours. The large variety
of equipment, such as transfusion tubing, blood warmer,
heparinized syringes, and an accurate machine to obtain a
rapid hematocrit value, must be prepared and calibrated.
The fetus should be monitored with continuous fetal
monitoring for the ensuing 4 hours (both during the procedure
and immediately after because decelerations of the fetal heart
rate are common and must be managed cautiously).
suggested reading
1. Basu S, Kaur R, Kaur G, Jain S. Severe hemolytic disease of
newborn due to non Rh D Antibody. Indian J Pediatr. 2011.
2. Basu S, Kaur R, Kaur G. Hemolytic disease of the fetus and
newborn: Current trends and perspectives. Asian J Transfus
Sci. 2001;5:3-7.
3. Furukawa K, Nakajima T, Kogure T. Example of a women
with multiple intrauterine death due to anti M who delivered
a live child after plasmapheresis. Exp Clin Immunogenet.
1993;10:167-7.
4. Guidelines for blood grouping and antibody screening in the
antenatal and perinatal setting. Australian and New Zealand
Society of Blood Transfusion Ltd. 3rd edn. 2007.
5. Guidelines for blood grouping and red cell antibody testing
during pregnancy. Transfus Med. 1996;6:71-4.
6. Jeon H, Calhoun B, Pothiawala M, Herschel M, Baron BW.
Significant ABO haemolytic disease of the newborn in a
group B infant with a group A2 mother. Immunohematology.
2000;16:105-8.
7. Kennedy MS. Perinatal Issues in Transfusion Practice In
American Association of Blood Banks Technical Manual. 16th
64  Section 1  Clinical Hemotherapy
edn. American Association of Blood Banks, Bethesda: 2008. p.
633.
8. Marwaha N, Dhawan HK, Thakral B, Kaur R, Basu S, Parmar
V. Severe ABO hemolytic disease of the newborn with a
positive direct antiglobulin test. Indian J Pathol Microbiol.
2009;52:292.
9. Moise KJ Jr. Non anti-D antibodies in red cell alloimmunisa­
tion. Eur J Obstet Gynecol Reprod Biol. 2000;92:75-81.
10. Mollison PL, Engelfret CP, Contreras M. Hemolytic disease
of the fetus and the newborn. In: Mollison PL, Engelfret CP,
Contreras M, eds. Blood transfusion in clinical medicine, 11th
edn. Oxford Blackwell Science; 2005. p. 508.
11. Pepperell RJ, Barrie JU, Fliegner JR. Significance of red cell
irregular antibodies in the obstetric patient. Med J Aust.
1997;2:453-6.
12. Pollock JM, Bowman JM. Anti-Rh (D) subclasses and severity of
Rh haemolytic disease of the newborn. Vox Sang. 1990;59:176-9.
13. Prasad MR, Krugh D, Rossi KQ, O'Shaughnessy RW. Anti D in Rh
positive pregnancies. Am J Obstet Gynecol. 2006;195:1158-62.
14. Thakral B, Agrawal SK, Dhawan HK, Saluja K, Dutta S, Marwaha
N. First report from India of haemolytic disease of the newborn
by anti c and anti E in Rh (D) positive mothers. Haematology.
2007;12:377-80.
15. Tondon R, Kataria R, Chaudhry R. Anti - M: Report of two cases
and review of literature. Asian J Transfus Sci. 2008;2:81-3.
16. Toy PT, Reid ME, Papenfus L, Yeap HH, Black D. Prevalence
of ABO maternal-infant incompatibility in Asians, Blacks,
Hispanics and Caucasians. Vox Sang. 1988;54:181-3.
17. Voak D, Mitcheli R, Bowell P, Letsky E, de Silva M, Whittle M.
Guidelines for Blood grouping and red cell antibody testing
during pregnancy. Transfus Med. 1996;6:71-4.
18. Walker RH. Relevance in the selection of serologic tests for the
obstetric patient. In: Garratty G, ed. Hemolytic disease of the
newborn. Arlington, VA: American Association of Blood Banks;
1984. p. 173.
19. Weinstein L. Irregular antibodies causing haemolytic disease
of newborn: a continuing problem. Clin Obstet Gynaecol.
1982;25:321-32.
Antenatal Screening of
Thalassemia
Reena Das
Hemoglobin (Hb) is a fascinating molecule which binds
oxygen in the lungs and delivers it in a precise and orderly
manner to the tissues. Human Hb consist of four heme groups
and four globin chains (2 α and 2 globin chains). Normal
human adults have predominantly (98%) adult Hb (ΗbΑ;
α2β2) and small (<2%) amounts of HbA2 (α2δ2,) and fetal Hb
(HbF; α2γ2) of <1%.1 Reduced synthesis of one or the other
type of globin chains results in decreased hemoglobinization
of the erythroid cells and an excess of the non-affected chains
which continue being synthesized normally, but cannot be
effectively removed and become harmful for the cell. This
group is known under the general term “thalassemia”, further
subdivided in alpha– and beta- form, according to the chain
whose synthesis is reduced. Defects in the structure of globin
chains are called Hb variants or hemoglobinopathies.2
Due to the autosomal recessive pattern of inheritance of
beta thalassemias, both the parents of an affected child with
thalassemia major (TM) are obligate carriers of one defective
beta globin chain and are known as β thalassemia trait (β
TT). Individuals with βTT are apparently normal and being
unaware that they are carriers, they constitute the reservoir
of the disease. The diagnostic hallmark to identify a βTT
is by quantitation of HbA2. Patients with a clinical severity
intermediate between βTT and TM are called thalassemia
intermedia (TI) and they show marked clinical and molecular
heterogeneity. Patients with TM present in infancy with
progressive pallor and hepatosplenomegaly and require
frequent three weekly intervals of blood transfusion to
sustain a reasonable quality of life. If not treated with regular
blood transfusions patients with TM die within their first few
years. Also children who receive regular blood transfusions
develop marked secondary iron overload and require regular
chelation therapy to reduce the deposition of siderotic iron.
The cost of treatment of a child with TM is approximately Rs.
1.5 lakh/year. Lifelong treatment is expensive and causes
immense financial strain to the patient and the family. Bone
Marrow transplant from a HLA matched sibling is the only
cure and costs Rs 7–10 lakhs.3 Therefore, screening for βTT
and antenatal diagnosis and selective termination of affected
19
fetuses after appropriate genetic counseling, being much
cheaper, is a cost effective measure to control TM.4
Since most marriages in India are arranged by the parents,
marriages are prevalent within the same castes and this leads
to higher frequency of thalassemia in certain communities.
Preventive strategies include a) Population screening at either
premarital or preconception stage, b) genetic counseling and
c) prenatal diagnosis. Earlier screening for βTT was carried
out in schools and colleges for determining the prevalence
of βTT in the general population. However, following up
with genetic counseling does not yield productive result
since many years may lapse between conveying the report
and planning the birth of their child. Presently a consensus
has evolved that screening the antenatal women during the
first antenatal visit is the most cost effective measure of
screening the population. All women who are found to be
carriers are advised to get their spouse screened. This strategy
has been found to be most effective as the concern to have a
normal healthy baby is a universal phenomenon. The other
group where targeted screening yields good return is ‘cascade
or extended family screening’ in family members who have
an index case of thalassemia major or severe thalassemia
intermedia. This subgroup is also initiated to the process of
screening and the frequency of finding βTT’s is much higher
than the general population screening.5
We are indeed fortunate that a screening strategy exists
by way of a simple blood test to identify individuals who are
carriers of a grave genetic disorder such as beta thalassemia.
A through hematologic evaluation including complete blood
count (CBC) using automated blood cell counters, which are
readily available, can be used as a basic screening strategy.
Almost universally all individuals who are βTTs show
hypochromic microcytosis (usually MCV will be <80 fl and
MCH <27 pg) with relative erythrocytosis (increased RBC
counts) and this is used to screen the population or specific
target groups such as antenatal women. Since the availability
of cation exchange high performance liquid chromatography
(HPLC) from BioRad, this has become the screening method
to analyze the HPLC pattern due to the speed, convenience
66  Section 1  Clinical Hemotherapy
as well as accuracy of the results. Quantitation of HbA2 of ≥4%
is the diagnostic hallmark to identify a βTT. Any individual
identified to be a βTT should be advised to get a screening
done of the partner before conception of a child. In the event
of finding any abnormal peak on the HPLC, a hemoglobin
electrophoresis should be carried out before reporting the
result of HPLC. This is due to the fact that many abnormal
peaks can be found in the same location such as Hb D Iran and
Hb E overlap in the region of HbA2 and Hb electrophoresis
is required to differentiate the two conditions. Though the
regional prevalence’s of the abnormal hemoglobins are
segregated, the population migration as well as inter caste
marriages is blurring such differences.6 DNA analysis for
specific beta gene mutations is commonly carried out by
either ARMS-PCR or by Reverse Dot Blot analysis. Many of
the specific abnormal hemoglobin analysis are carried out by
specific PCR-RFLP such as Hb D Punjab, Hb E, Hb S and Hb
O Arab.7
More than 2200 children with TM are on a regular blood
transfusion program in the Punjab region and unaccounted
for number of affected children with TM have died due to the
complications of the disease. The department of Hematology
at PGIMER, Chandigarh has been providing diagnostic
support to the patients of the region since 1993 and holistic
management of children with TM is provided by the Hemato-
oncology unit, Pediatrics department. Since the last five years,
with the help of the Obstetrics and Gynecology department
routine complete screening (comprising of automated blood
cell counts and HPLC for Hb subtyptes) is being provided
to all antenatal women referred to the department. Prenatal
diagnosis has been carried out on over 700 pregnancies in the
region. Many transfusion centers such as those in PGIMER,
Chandigarh; GMCH, Sector 32, Chandigarh; GMCH, Patiala
and GMCH, Amritsar are among the many centers in Punjab
who are committed to provide safe blood to the patients with
TM and TI. Due to the marked health burden to our limited
health resources and cost involved only 10% of the patients are
being optimally treated with regular blood transfusions with
safe blood and iron chelation in India. Therefore, aggressive
preventive strategies are needed to be taken to decrease the
prevalence of TM.
We have been involved in prenatal diagnosis of >700
cases by using ARMS-PCR, RFLP and DNA sequencing. The
problems encountered are occasional non-amplification
of primers. Most centers are performing VNTRs to exclude
maternal contamination. Though our country has the
expertise and technical knowhow and many more centers
throughout the country are required to tackle the burden
of the disease. Offering prenatal diagnostic services is the
most effective way of controlling thalassemia. The problems
encountered in our country include, a vast population, no
national screening program, unawareness amongst the
medical fraternity and shortage of trained centers and staff.
Many patients present to the obstetricians and pediatricians
late in their pregnancy to work up for the antenatal testing.
(Unpublished data) A few subjects are ‘silent’ carriers who
pose to be diagnostic difficulties in screening programs and
it is important to keep this fact in mind during the process of
genetic counseling.8
References
1. Higgs DR, Thein SL, Wood WG. Human haemoglobin. In:
Weatherall DJ, Clegg B, eds. The thalassaemia syndromes. 4th
edn. Oxford, England: Blackwell Science, 2001.
2. Weatherall DJ, Clegg JB, editors. The thalassemia syndromes.
4th edn. Oxford: Blackwell Science, 2001.
3. Verma IC, Bijarnia S. The burden of genetic disorders in India
and a framework for community control. Community Genetics.
2002;5:192-6.
4. Garewal G, Das R. Spectrum of β thalassemia mutations
in north India. International Journal of Human Genetics.
2003;3:217-9.
5. Garewal G, Das R, Kaur J, Marwaha R K, Gupta I. Establishment
of Prenatal Diagnosis for β-Thalassemia: A step towards its
Control in a Developing Country. Annals of Human Biology.
2005;32(2):138-44.
6. Madan N, Sharma S, Sood SK, Colah R, Bhatia HM. Frequency
of b-thalassemia and other hemoglobinopathies in India.
Indian Journal of Human Genetics. 2010;16:16-25.
7. Colah RB, Gorakshakar AC, Nadkarni AH. Invasive
and non-invasive approaches of prenatal diagnosis of
haemoglobinopathies: Experiences from India. Indian Journal
of Medical Research. 2011;134:552-60.
8. Garewal G, Das R, Awasthi A, Ahluwalia J, Varma N. Marwaha
RK. The clinical significance of the spectrum of interactions of
CAP+1 (A®C), a silent beta globin gene mutation, with other
beta thalassemia mutations and globin gene modifiers in
North Indians. Eur J Haematol. 2007;79(5):417-21.
Neonatal Transfusion Practice
A Surekha Devi
Sick neonates are one of the most heavily transfused groups of
patients in modern medicine. The transfusion requirements
of the neonate are recognized as unique. Like any therapeutic
decision, neonatologists should decide to transfuse based on
the risks, benefits and alternatives to transfusion. Recipients
run the risk of both over-transfusion and under-transfusion.
Administration of blood components is done on a per weight
basis. Guidelines are derived from consensus, with several of
them demonstrating appropriateness in published studies.
Blood and blood component specifications:
General recommendations (fetuses,
neonates, infants and children)
Donors
Components to children under 1 year of age must be prepared
from blood donated by donors who have given at least one
previous donation within the past 2 years, which was negative
for all mandatory microbiological markers.
Special Needs
Leukocyte depletion: All components other than granulo­
cytes concentrates should be leukocyte depleted (not more
than 5 × 106 leukocytes per unit) at the time of manufacture.
Cytomegalovirus (CMV): The guidelines of the UK trans­
fusion services state that blood transfused in the first year of
life should be CMV negative. Fetuses, infants weighing under
1.5 kg, immunodeficient patients and stem cell transplant
recipients are at risk of transfusion transmitted CMV. In an
emergency, where CMV-seronegative blood components are
not available, transfusion of leukodep­leted components is an
acceptable.
Irradiation: Cellular blood components are irradiated to
a dose of 25 Gy. For IUT and large volume transfusion, e.g.,
exchange transfusion, the component should be used within
24 hours of irradiation and within 5 days of donation. Red cells
20
for top-up transfusion may be irradiated at any time up to 14
days after collection, and stored for 14 days from irradiation.
Platelets transfused in utero to treat alloimmune throm­
bocytopenia and platelet transfusions given after birth
to infants who have received either red cells or platelets
in utero should be irradiated.
All granulocytes should be irradiated for patients of any
age and transfused as soon as possible after irradiation.
Indications of Irradiation of Cellular
Blood Components
• Intrauterine transfusions (IUT)
• Exchange transfusion (ET) of red cells
• Top-up transfusions after IUT
• Directed donations from a first- or second-degree relative
• Immunodeficiency
Plasma and Platelet Compatibility
Platelets should be ABO and RhD identical with the recipient,
if not available compatible components lacking high titer
anti-A or anti-B should be transfused to group A or B
recipients.
Group AB FFP is recommended for transfusion in the first
year of life.
Administration
All components should be transfused through a standard
blood giving set with a screen filter (170–220 microns). Where
small volumes are drawn into a syringe an appropriate filter
must be used. Microaggre­grate filters are not required for
leukoreduced compo­nents.
Pretransfusion Testing
Samples from both mother and neonate should be obtained
for initial ABO and RhD group determination.
68  Section 1  Clinical Hemotherapy
Investigations on Maternal Sample
• ABO and RhD group
• Screen for the presence of atypical red cell antibodies
Investigations on Neonate Sample
• ABO and RhD. ABO by cell group only
• Direct antiglobulin test (DAT)
• In the absence of maternal serum, screen neonate’s serum
for atypical antibodies by an indirect antiglobulin test
(IAT)
A positive DAT on the neonate’s red cells or an atypical red
cell antibody in maternal or neonatal serum suggests possible
hemolytic disease of newborn (HDN).
Selection of Blood Component
Components should be
• Of the neonate’s own ABO and RhD group, or an alternative
compatible ABO and RhD group.
• Compatible with any ABO or atypical red cell antibody
present in the maternal or neonatal plasma.
• Neonatal samples are not included in electronic cross-
match protocols as maternal serum is used for cross­match.
• Small volume transfusions can be given repeatedly over
the first four months of life without further serological
testing, provided there are no atypical maternal red cell
antibodies in infant’s serum and infant is DAT negative.
• If either the antibody screen and DAT are positive,
compatibility testing will be necessary.
• After the postnatal age of 4 months, compatibility test is
conducted as in adult practice.
Intrauterine Transfusion
Intrauterine red cell transfusion is indicated to correct fetal
anemia caused by red cell alloimmunization (RhD, Rhc and
K), or for fetal parvovirus infection. Intrauterine platelet
transfusions are indicated to correct fetal thrombocytopenia
caused by platelet alloimmunization. Aims of IUT are
• To prevent or treat fetal hydrops before the fetus can be
delivered
• To enable the pregnancy to advance to a gestational age
that will ensure survival of the neonate
– Starting transfusion as late and safely possible but
before hydrops develops
– Maximizing the intervals between transfusion by
transfusing large volume of red cells
Red Cell Preparations
• Be group O or ABO identical with fetus, RhD negative,
K-negative. In case of hemolysis due to maternal anti-c,
RhD positive, c-negative blood is given
• Be cross-match compatible with maternal serum and
negative for relevant antigen(s) determined by maternal
antibody screen
• Be <5 days old in CPD anticoagulant
• Be CMV seronegative
• Be irradiated
• Have a hematocrit of up to 0.75
• Blood is warmed with care, temperature not exceeding
30°C
• Volume is calculated from the formula of Rodeck and
Deans:
Desired PCV – Fetal PCV
× Fetoplacental blood volume
Donor PCV – Desired PCV
• Be transfused at a rate of 5–10 mL/min
Platelet Transfusions
• Be group O RhD negative and have low titer anti-A or
anti-B
• Be HPA compatible with maternal antibody
• Collected by apheresis
• Be irradiated
• Be concentrated to a platelet count of 2000 × 109/L
• Be in a volume calculated from formula
Desired platelet increment
× Fetoplacental blood volume
Platelet count of concentrate
• Be transfused at a rate of 1–5 mL/min
Compatible platelets should be available at the time of
diagnostic fetal sampling to prevent fetal hemorrhage. Teflon
coated needles should be used.
Transfusions of preterm infants
• All NICUs should have written, consensus based guidelines
that underline transfusion policy. Guidelines should be
regularly updated in-line with the best available evidence
and their implementation should be regularly assessed by
audit.
• Most preterm infants with birth weight of less than 1000 g
will require RBC transfusion, majority of infants with birth
weight greater than 1500 g do not require RBC transfusion.
• Minimizing donor exposure is important in reducing the
risk of transfusion-transmitted infections. Infant who is
likely to require recurrent RBC transfusion should have
pedipack assigned to them.
• Blood loss due to repeated phlebotomy is important cause
of anemia of prematurity requiring transfusion support in
low birth infants.
• The aim of top up transfusion is to restore or maintain
adequate tissue oxygen delivery without a marked increase
in oxygen consumption.

• Erythropoietin (EPO) cannot be recommended as the re­
cent Cochrane Database meta-analysis (2005) showed an
increased risk of retinopathy of prematurity with early
EPO treatment.
Neonatal transfusions
Exchange Transfusion (ET)
Exchange transfusion is used to manage severe anemia
at birth, in the presence of heart failure and to treat severe
hyperbilirubinemia, caused by HDN.
Aim is to remove both the antibody-coated red cells
and the excess bilirubin. It should be done by staff who are
experienced in the procedure.
Single volume exchange: Exchanging the estimated
volume of the baby’s blood in a single volume exchange will
remove 75% of red cells.
Double volume exchange (160–200 mL/kg) removes 50%
of available intravascular bilirubin.
Component and Procedure Specification
• Be group O or ABO compatible with maternal and neonatal
plasma, RhD negative (or RhD identical with neonate).
• Be negative for any red cell antigens to which the mother
has antibodies.
• Be IAT-cross-match compatible with maternal plasma.
• Be 5 days old or less.
• Be collected into CPD anticoagulant.
• Be CMV seronegative.
• Be irradiated and transfused within 24 hours of transfus­
ion. Irradiation is essential if the infant has had a previous
IUT and is recommended for all ETs.
• Have a hematocrit of 0.50–0.60.
• To warm the blood product prior to transfusion.
• Volume transfused is 80–160 mL/kg for a term infant and
100–200 mL/kg for a preterm infant.
ABO Hemolytic Disease of the Newborn (HDN)
• Group O blood compatible with the maternal plasma,
should be used for transfusion.
• If an ET is required in ABO HDN, group O red cells with
low titer plasma anti-A and anti-B, or with group O red
cells suspended in AB plasma should be used.
Red Cells for Top-up Transfusions
in Neonates
• Be ABO compatible with mother and infant, and infant’s
RhD group.
• Be IAT compatible with maternal plasma (if available) or
neonate’s plasma for first transfusion (and subsequent
Chapter 20  Neonatal Transfusion Practice  69
transfusions up to four postnatal months if atypical mater­
nal antibodies present).
• Be 35 days old or less (if in SAG-M or similar additive
solution) or 28 days old or less (if in CPD).
• Have a hematocrit of 0.50–0.70.
• Be irradiated if appropriate.
• Be infused in a volume of 10–20 mL/kg.
• Be aliquotted donations (pedipak) from a single unit dedi­
cated to one infant.
Guidelines for Administration of Red Cells
• Transfusion thresholds for red cell transfusion
− Anemia in the first 24 hour—Hb 12 g/dL
− Acute blood loss/cumulative blood loss in 1 week—10%
BV
− Neonate receiving intensive care—Hb 12 g/dL
− Chronic oxygen dependency—Hb 11 g/dL
− Late anemia, stable patient—Hb 7 g/dL
• Surrogate markers of anemia include respiratory irregu­
larity, tachycardia, poor weight gain, lethargy, poor suck
and increased blood lactate levels.
• Patients with higher oxygen extraction ratio (>40%)
Platelets for Neonatal Transfusions
• Be ABO identical or compatible, Rh identical or compatible.
• Be HPA compatible in infants with alloimmune thrombo­
cytopenia.
• Be produced by standard techniques (plateletpheresis).
• Be irradiated if appropriate.
• Be infused in a volume of 10–20 mL/kg.
• Administration of platelets:
− Preterm or term neonate, with bleeding—50 × 109/L
− Sick preterm or term neonate, not bleeding—30 ×
109/L
− Stable preterm or term neonate, not bleeding—20 ×
109/L
Fresh Frozen Plasma for Neonatal
Transfusions
• Be group AB, or compatible with recipient’s ABO red cell
antigens.
• Be infused in a volume of 10–20 mL/kg.
• Virus inactivated plasma should be used for treatment of
patients with inherited coagulation deficiencies.
Cryoprecipitate for Neonatal Transfusions
Common indication for cryoprecipitate is to restore fibrinogen
levels in patients with acquired hypofibrinogenemia in DIC
and massive transfusions.
• Should be transfused only ABO-compatible cryoprecipi­tate.
70  Section 1  Clinical Hemotherapy
• Cryoprecipitate separated from AB plasma can also be
given
• Dose is 5 mL/kg which raises fibrinogen level to about
0.6–1.0 g/L
Granulocytes for Neonatal Transfusions
• Be ABO and RhD compatible with the recipient.
• Dose is 1–2 × 109 granulocytes/kg.
• Irradiated to a dose of 25 Gy prior to administration.
• Be CMV seronegative.
• Recommended two or more daily infusions of an appro­
priate dose.
Transfusions in necrotizing
enterocolitis (NEC)
• Be transfused with saline washed red cells or red cells in
SAG-M as this is relatively plasma-free saline washed red
cells or
• ET may be necessary if a patient with NEC develops he­
molysis, and T-activation is likely cause.
• Platelets, FFP and cryoprecipitate should be administered
only when clearly indicated.
Transfusions in Extracorporeal
membrane oxygenation (ECMO)
This is highly specialized respiratory support, infants are
anticoagulated with heparin, and require regular monitoring
of coagulation parameters and platelet count.
• Following the initial ‘coating’ prime with albumin, priming
with whole blood, packed red cells or packed cells and FFP
may be indicated
• Blood should be as fresh as possible not >5 days old
• Whole blood contains a significant amount of relatively
fresh plasma containing all coagulation factors other than
FVIII and FV
• Red cells in additive solution are not advised for priming
• Platelet transfusions are given to maintain platelet count
above 100 × 109/L and FFP given to manage excessive
bleeding.
• Fibrinogen level should be maintained above 0.8–1.0 g/L
with cryoprecipitate 5 mL/kg.
• Antithrombin infusion is given to keep the levels adequate
for heparin function.
• A normal hematocrit (of around 0.45) has been associated
with increased risk of clotting in the circuit, which may be
reduced by lowering the hematocrit to 0.35.
Neonates are the longest living survivors of blood trans­
fusion and are at risk for the long-term consequences of
transfusion transmitted infections. Every effort has to be
made to reduce transfusions and donor exposure in this most
vulnerable group of transfusion recipients.
suggested reading
1. JMO’ Riordon, et al. Transfusion of blood components to
infants under four months: Review and Guidelines. Irish Medi­
cal Journal Supplement June 2007, Volume 100, Number 6.
2. NA Murray, IAG Roberts. Neonatal transfusion practice. Arch
Dis Child Fetal Neonatal Ed.2004; 89:F101-F107.
3. Neonatal transfusion recommendations at RCH, Melbourne,
Australia. Contents authorized by Webmaster. Last updated
12 Dec, 2012.
4. Strauss RG. Current issues in neonatal transfusion. Vox Sang.
1986;51(1):1-9.
5. Susan D.Roseff. Pediatric transfusion. A physcian’s Handbook.
2nd edn. 2006. AABB, Bethesda, Maryland, USA.
6. Transfusion guidelines for neonates and older children.
Blackwell Publishing, British Journal of Haematology. 2004;
124:433-53.
 Transfusion Practice in Transplantation
Transfusion Practice in
Bone Marrow Transplantation
and HLA Matching
M Joseph John
Hematopoietic stem cell transplantation (HSCT) is today
widely used to treat hematologic, immune and genetic
diseases. The annual rate of allogenic bone marrow trans­
plants now exceeds 60,000 world wide and more than 1200
in India [as per the ISCTR data (Indian Stem Cell Transplant
Registry data)]. In thalassemia, BMT now provides a cost
effective alternative to transfusion/chelation therapy with
long-term transfusion and iron overload free life.
Hematopoietic stem cells (HSCs) have the potential of
indefinite self renewal, proliferation and differentiation into
different cell lineages. Stem cells are found in the bone mar­
row, cord blood and a small number in the peripheral blood.
HLA Typing
Major histocompatibility complex (MHC) is located in the
short arm of chromosome 6. Human leukocyte antigens are
the gene products of MHC. They are all cell surface antigens.
Class I: HLA A, B and C. They are located farthest from the
centromere. Present in all nucleated cells except mature
trophoblast and neural tissue.
Class II: HLA, D-DR, DP and DQ. Located closest to the
centromere. Present on B lymphocytes, monocytes, macro­
phages, dendritic cells, endothelial cells, Langerhans cells
and activated T lymphocytes.
Class III: Serum proteins, complement, TNF, properidin.
They separate Class I and II in their location.
Function of HLA: HLA antigens restrict and therefore regulate
the immune response in a highly specific way. The function of
antigen presenting cells, is to incorporate antigen, to process
it, and to present it either intact or fragmented in association
with self HLA so that it can be recognized by T-lymphocytes.
This dual recognition generates cytochemical signaling which
results in the proliferation of cells subsets of T-lymphocytes,
activated cytotoxic T-lymphocytes subsequently attack all
cells expressing self HLA\foreign antigen combination of their
surface. This is an efficient mechanism for destroying viral
21
infected cells. Activated helper T-cells stimulates appropriate
B-lymphocytes to produce an antibody.
These antigens determine whether an organ would be
accepted or rejected when transplanted into another indivi­
dual.
HLA typing can be done by serological or DNA PCR based
methods. Of late the PCR based test is being done. Before the
availability of DNA based 10 antigen typing was available, 6
antigen typing with serological method was being done for
AB and DR. However, now with the 10 antigen typing HLA-A,
B, C, DR and DQ up to the allelic resolution can be done. This
helps in choosing the most appropriate family donor or a
matched unrelated donor.
HSCT differs from many other solid organ transplant for
the fact that it can be done across the blood groups even for
major, minor and major-minor mismatches. This is essentially
because blood group antigens are not expressed on the stem
cells and once the engraftment occurs, the donor stem cells
will produce the red blood cells of the donor blood group. In
short if a B group patient is transplanted with A group, post-
transplant that person would become A group.
Transfusion Practices
It is important to consider certain principles and transfusion
practices to minimize the complications related to trans­
fusion of blood components in the peri-transplant period.
Hematopoietic stem cell transplant (HSCT) patients often
require intensive blood component support. Transfusion
may be complicated by transfusion transmitted infection
(TTI)—both viral and bacterial, transfusion-associated
(TA)-GvHD, febrile non-hemolytic transfusion reactions
(FNHTR) and transfusion-related acute lung injury (TRALI).
Alloimmunization (AI) to red cell antigens may cause
difficulties in selecting compatible blood while AI to the
human leukocyte antigens (HLA) present on platelets may
cause refractoriness to subsequent transfusions of randomly-
selected platelets.
72  Section 1  Clinical Hemotherapy

Key Points to Remember while Transfusing
the Blood in a Transplant Patient (Pre or Post)
• Standard blood bank practices
• Use a leukodepletion filter
• Irradiate the blood before transfusion
• Choosing the appropriate product in case of incompati­
bility.
Importance of Using Leukodepleted Products
Transfused leukocytes cause alloimmunization (Table 1) to
HLA Class 1 antigens (HLA AI) in a proportion of patients. This
may be manifested clinically as febrile non-hemolytic
transfusion reactions (FNHTR), although these may also
be caused by antibodies to neutrophils, platelets or plasma
proteins and cytokines which accumulate in stored blood
components, especially packed red blood cells.
is currently the recommended method for TA-GVHD
prevention. Leukodepletion by current filtration technology
is inadequate for this purpose.
TA-GVHD occurs when donor lymphocytes from
transfused blood engraft in the recipient and mount an
immune response. There appears to be a particular risk
when donor and recipient share a particular haplotype,
as occurs within families or in populations with restricted
haplotypes. Under certain circumstances, these cells engraft
and proliferate in the patient. Interaction between donor T
lymphocytes and recipient cells carrying either class I or II
HLA antigens results in cellular damage which may be natural
killer (NK) cell mediated. The risk is highest in patients who
are immunosuppressed. Typically TA-GVHD occurs 7–14
days post transfusion with clinical features of fever, skin
rash, hepatitis, diarrhea and pancytopenia. Although the
incidence is approximately 1%, it is fatal in more than 90%
patients.

Gamma-irradiation of Blood Components and Blood Products which Requires Irradiation

Transfusion Associated-Graft Versus Host All cellular blood products where leukocyte contamination is
Disease (TA-GVHD) present.
Blood irradiation is a process by which the proliferative • Packed or whole blood
potential of viable T lymphocytes contaminating the cellular • Random donor platelets (RDPs) and single donor platelets
blood product is destroyed. This can be done by using gamma (SDPs).
irradiation or ultraviolet light. • Granulocytes.
As per the BCSH Task force, the minimum dose achieved The best time to irradiate is at the time of release of the
in irradiation field should be 25 Gy with no part receiving blood product. However, platelets can be irradiated and kept
more than 50 Gy. for up to 5 days.

Rationale of Irradiation Indications

To prevent transfusion associated graft-versus host disease
(TA-GVHD). As per BCSH task force, gamma irradiation
Table 1: Adverse effects of transfused white blood cells
HLA alloimmunization •  FNHTR
•  Refractoriness to random
donor platelets
•  Graft rejection
•  Shortened red cell survival
Transmission of microorganisms •  CMV
•  HTLV-1/11
•  Toxoplasma gondii
•  Yersinia enterocolitica
Immunomodulation •  GVHD
•  Activation of viruses in host
cells, e.g. HIV-1
•  Immune suppression of T- and
NK-cell functions
Affecting the quality of stored •  Microaggregate formation
blood •  Metabolic deterioration
during storage
• Allo-HSC recipients from time of conditioning therapy for
6 months or until the lymphocyte count is 1 × 109/L in the
absence of GvHD
• Allo-HSC donors if they were to receive blood
• Auto-HSC recipients (from 7 days before harvest until 3
months post-transplant)
• All donations from HLA-matched donors or 1st or 2nd
degree relatives
• All patients with Hodgkin disease at any stage of therapy
• All patients treated with purine analogs, e.g. fludarabine
• All patients with congenital immunodeficiency states
Donor/Recipient ABO Incompatibility and
Transfusion Support
Approximately 15–25% of HLA identical sibling donor/
recipient pairs are ABO incompatible. The figure is higher in
alternative donor transplants with almost 40%. .
In myeloablative transplants, ABO incompatibility is
associated with an increased risk of delayed red cell engraft­
ment, pure red cell aplasia (PRCA), hemolysis, TTP and
increased transfusion requirements.
 Chapter 21  Transfusion Practice in Bone Marrow Transplantation and HLA Matching  73
Definitions
Major ABO incompatibility is defined as the presence in
the recipient’s plasma of anti-A, -B or -A, B alloagglutinins
reactive with the donor’s red cells, e.g. donor group A and
recipient group O.
Minor ABO incompatibility is defined as the presence of
anti-A, -B or -A, B alloagglutinins in the donors plasma
reactive with the recipient’s red cells, e.g. donor group O and
recipient group A.
Major plus minor ABO (bidirectional) incompatibility is
defined as the presence in both the donor and recipients
plasma of anti-A, -B or -A, B alloagglutinins reactive with
recipient and donor cells respectively, e.g. donor group A and
recipient group B.
Incompatible Stem Cell Graft Infusion
If the alloagglutinin titer is less than 1:64 unmodified bone
marrow or PBSC grafts may be infused. At higher titers red
cells should be removed from the graft.
Plasma may be removed from the transplant in cases of
minor ABO mismatch where the anti-A titer is high to avoid
acute hemolysis in the recipient.
Delayed hemolytic transfusion reactions may follow the
infusion of donor HSC where there is a minor ABO mismatch.
This is called passenger lymphocyte syndrome (PLS) and
occurs because of a secondary (anamnestic) immune res­
ponse mediated via memory B-cells in the graft against
recipient ABO antigens. A rise in anti-A or B titer is seen
together with anemia and jaundice.
ABO Incompatibility in BMT:
Graft Management
Major ABO Incompatibility
Manage by RBC reduction of graft
• < 20–30 mL donor prbc is considered safe
• Recipient anti-A or B titers (both IgM and IgG) ≤ 1:16 is
considered safe
• Typically a problem for marrow, not PBSC
• RBC reduction (e.g. by sedimentation) may lose 20–30%
of mononuclear cell (MNC) and CD34
• Recipient plasmapheresis used by some if dose is limiting
Minor ABO Incompatibility
• Manage by plasma reduction of graft
• Applies to both marrow and PBSC
• < 5 mL donor plasma/kg is considered safe, OR
• If donor anti-A and/or –B titer ≤ 1:128—no plasma
reduction
Major Incompatibility: Transfusion
Management
Hemolysis of Nascent Engrafted rbc
(10–15% of cases; e.g. A to O)
• Hemolysis 1–3 weeks after transplant and may last for 6
months.
• 40% - positive DAT; < 10% have significant hemolysis
• If high pre-BMT ABO titers (>1:16); Cyclosporin for GVHD
Delayed rbc Engraftment—up to 6 months or more
• Rarely delayed neutrophil or platelet engraftment
Diagnosis of Hemolysis
• Hemolysis parameters—Hb, LDH, bilirubin
• Positive DAT and anti-donor ABO antibody on rbc +/-
plasma
Management
• Test periodically for disappearance of recipient anti-donor
Ab by Coombs test (recipient plasma and donor type [A or
B] rbc)
• Give recipient type rbc until anti-donor Ab by ICT is
negative for at least 2 times.
• Manage hemolysis similar to delayed hemolytic transfu­
sion reactions (DHTR) (hydration; diuretic)
Example
Major ABO incompatibility
• Recipient is type O and •  Donor is type A, B, or AB
• Graft (prevent acute hemolysis):
– Limit rbc to 20 mL OR only if recipient anti-A, -B titer
is > 1:16
• Transfusion (prevent delayed hemolysis):
– 2 phases—before vs after disappearance of recipient
anti-donor Ab
– Initially transfuse with recipient type rbc (or O)
– Check for isohemagglutinins by ICT ~ weekly until gone
– When isoagglutinins gone × 2; safe to give donor type
rbc
– Recipient isoagglutinins may persist for > 6 months,
resulting in “pure rbc aplasia”; especially if non-
myeloablative transplant
– Plasma products should be donor type (e.g. A) or AB
Minor-side ABO incompatibility
Recipient – A, B, AB
• rbc incompatible with donor plasma Abs
• Plasma compatible with donor rbc
74  Section 1  Clinical Hemotherapy
Donor – O
• Plasma – anti-A, -B antibodies
• rbc – compatible with all
Problems
• Immediate hemolysis of recipient rbc due to plasma in
graft
• Delayed hemolysis of recipient rbc due to Passenger
lymphocyte syndrome (PLS)
Minor Incompatibility: Passenger
Lymphocyte Syndrome
• Delayed hemolysis of recipient rbc (10–15% of cases; e.g.
O to A)
– Hemolysis 5 – 21 days after transplantation
• Due to “passenger lymphocyte syndrome” (PLS)
• Donor B cells produce anti-recipient ABO antibody
– Hemolysis in 15–75%; severe in 3%; ? increased GVHD
– Increased risk in nonmyeloablative transplant, no
Methotrexate (MTX), PBSC, and T-cell depleted BMT,
increased antibody titer in the donor.
– Anti-recipient ABO ishohemagglutinin disappears
within one year in most patient.
• Diagnosis of hemolysis:
– ↓ Hb, Hp; ↑ LDH, and bilirubin.
– +DCT; anti-recipient ABO isohemagglutinin on rbc
(eluate)
– Anti-recipient ABO isohemagglutinin in plasma
Risk of Hemolysis in PLS
• No transfusion with donor type rbc before transplant.
• High patient hemoglobin – no rbc transfusion required
• PBSC graft (10 × more B lymphocytes than marrow)
• No MTX for GVHD prevention
• Conditioning (worse in nonablative transplant),
• High donor antibody titer
• T-cell depletion (no T-regs)
Management of PLS
• Prevention
– Transfuse with donor/type O rbc early, before
transplant, to minimize residual “recipient” type rbc
left to hemolyse
– Exchange transfusion may be indicated for high risk
patient.
• Monitor after transplant
– Hemolysis—daily DCT, LDH, Hb (×2 daily) from day +5
to +14
– Donor antibody – q3 days for anti-recipient ABO Ab by
Coombs test.
• Manage hemolysis similar to acute or delayed HTR
– Hydrate; urine flow 100 cc/hr +/- lasix, NaHCO3.
– Transfuse with donor type rbc or type O rbc.
– Exchange transfusion may be indicated.
Example: Minor ABO Incompatibility
• Recipient is type A (or B or AB)
• Donor is type O
• Graft (prevent acute hemolysis)
– Limit plasma to 5 mL/kg OR only if donor anti-A, -B
titer is > 1:128
• Transfusion (prevent delayed hemolysis)
– Two phases – (1) early reduction of recipient rbc;
(2) emergence of donor Ab
– Switch to transfusion with donor type rbc (or O) ASAP
before transplant
– Post-transplant d+4 – d+14, check
twice daily Hb/Hct
Daily DCT; LDH; +/- Indirect bilirubin;
– ABO type q3d - Presence of residual recipient (e.g. A)
rbc and Emergence of donor type (anti-A) isoaggluti­
nin.
– Consider early exchange transfusion with donor (type
O) rbc if high risk – i.e. mostly host type rbc and/or
NST and/or PBSC graft and/or no methotrexate used
– Plasma products should be recipient type (A) or AB
until
• Donor hematopoiesis established and
• No residual recipient rbc × 2
Example: Bidirectional ABO Incompatibility:
Recipient is type A and Donor is type B
• Graft (prevent acute hemolysis)
– RBC reduce to < 20 mL rbc
– Plasma reduce to < 5 mL plasma/kg
• Transfusion (prevent delayed hemolysis)
– Switch to transfusion with type O rbc ASAP PRE
transplant
– Monitor hemolysis post-transplant d+5 to d+14
• Hb/Hct x2/d; daily DAT; LDH; +/- indirect bilirubin;
• Donor anti-A/B isoagglutinin q 3 d
• Residual recipient rbc
– Consider early exchange transfusion with type O
rbc if host’s rbc are mostly host type and/or non
myeloablative transplant and/or PBPC graft and/or no
MTX used.
– Switch to donor type rbc when recipient anti-A/B is
gone (by ICT × 2)
– Plasma products should be AB or have low titer
isoagglutinin (i.e. < 1:100) until no residual recipient
rbc
Blood Groups Used for
Transfusion Support
Pre-transplant, recipient-type red cells and platelets should
be given (Table 2).
Post-transplant: For major ABO mismatch use group O red
cell products, irrespective of ABO group of recipient or donor
 Chapter 21  Transfusion Practice in Bone Marrow Transplantation and HLA Matching  75

Table 2: Selecting appropriate blood products for recipients of For minor ABO mismatch use red cells of the donor type,
ABO/Rh mismatched stem cell transplants i.e. group O throughout.
Give platelets and plasma of recipient type until recipient-
Donor Recipient Red cells Platelets FFP
type red cells are no longer detected.
Major ABO A O O A A For major and minor ABO mismatch use group O red
incompatibility B O O B B cells until recipient ABO antibodies are undetectable and the
antiglobulin test is negative and then switch to donor type.
AB O O AB AB
For platelets and plasma use group AB until recipient red
AB A O,A AB AB cells are undetectable.
AB B O,B AB AB Following graft rejection, revert to recipient-type red cells
Minor ABO O A O A A and platelets.
incompatibility O B O B B
O AB O AB AB
suggested reading
A AB O,A AB AB 1. EBMT Hand book on Stem cell transplantation. 2008. Chapter
7. Transfusion policy.
B AB O,B AB AB 2. Guidelines on gamma irradiation of blood components for the
Bidirectional ABO A B O AB AB prevention of transfusion associated graft versus host disease.
incompatibility BCSH Blood transfusion task force. Transfusion medicine,
B A O AB AB
1996;6:261-71.
3. James L Gajewski, et al. A review of transfusion practice before,
until recipient ABO antibodies are undetectable and the during, and after hematopoietic progenitor cell transplantation.
antiglobulin test is negative. Give platelets and plasma from Blood. 2008;112:3036-47.
donors of the recipient’s ABO type until recipient red cells are
no longer detected.
Transfusion Practice in Renal
Transplants Recipients
Vimarsh Raina, Aseem K Tiwari, Ravi Dara
The first successful renal transplantation in India was
performed on 2nd February 1971 by the team led by Dr Mohan
Rao (Urologist) and Dr KV Johny (Nephrologist) at Christian
Medical College, Vellore. Since then, the program of renal
transplantation has come a long way. Today many centers are
carrying out organ transplantation. The care of CRF patients
has improved due to the easy availability of hemo/peritoneal
dialysis. Better immunosuppressive drugs have reduced
the complications and improved graft and patient survival.
Transfusion medicine holds a place of prime importance
in organ transplant surgeries. The greatest advance in
clinical implementation of ABO-incompatible transplants
came about through desensitization and isoagglutinin
elimination techniques with immuno adsorption and anti-
CD20 antibodies becoming the norm. The implications and
practices of transfusion medicine in organ transplant are
also undergoing drastic changes. We have to understand
the immunological responses like auto-recognition, allo-
recognition and mechanism of rejections (acute and chronic)
before we can appreciate the effect of transfusion on renal
engraftment and ABO incompatible transplants.
Auto-recognition
Human leukocyte antigens (HLA) are a set of human major
histocompatibility complex derived glycoproteins that are
expressed on cell surface and allow for discrimination of self
antigens from non-self.1 HLA have been classified into two
major groups, Class I (HLA-A, HLA-B, and HLA-C) and Class
II (HLA-DP, HLA-DQ, and HLA-DR). Class I HLA molecules
are expressed on the surface of all nucleated cells and are
recognized by cytotoxic T cells (CD8+). Cytotoxic T cells
promote target cell destruction through apoptosis and release
of cytotoxic proteins. Class II HLA molecules are expressed
solely on the surfaces of antigen-presenting cells (APCs).
APCs include dendritic cells, macrophages and activated
B lymphocytes. APCs are vital in initiating the immune
response and stimulating helper T cells (CD4+). Some T
helper cells secrete cytokines that recruit cytotoxic T cells, B
lymphocytes or APCs, while others secrete cytokines which
attenuate the immune response. When a B lymphocyte binds
22
to an antigen and receives a cytokine signal from a helper
T cell, it can differentiate into a plasma cell or a memory B
cell. Plasma cells secrete antibodies which can destroy target
antigens while memory B cells cause long term immunity
and rapidly activate the immune system upon subsequent
exposure to the target antigen.1
Allo-recognition
Recognition of the antigens displayed by the transplanted
organ (alloantigen) is the prime event initiating the immune
response against an allograft.1
In the direct pathway, donor APCs migrate to the recipient’s
lymph nodes and present donor HLA glycoproteins to T cells.
In the indirect pathway, recipient APCs migrates into the
allograft and phagocytizes alloantigen. The HLA glycoproteins
are then presented to recipient T cells in the lymph nodes.
Regardless of whether the alloantigen is presented via the
direct or indirect pathway (referred to as Signal 1), a second
co-stimulatory signal (referred to as Signal 2) must also take
place for T cell activation. This is an interaction between one
of several co-stimulatory receptors and paired ligands on the
surfaces of APCs and T cells.1
Allograft Rejection
Hyperacute Rejection
Hyperacute rejection is an immediate recipient immune
response against an allograft due to preformed recipient
antibodies directed against the Donor’s HLA.1
Those at highest risk have HLA or ABO blood group
antibodies including patients with a history of previous organ
transplantation, multiple blood transfusions, multiparous
women and mothers receiving organs from their children.1,2
Acute Rejection
Acute rejection is also a cell mediated process that generally
occurs within 5 to 90 days after a transplant, although it can
occur after this time.1
 Chapter 22  Transfusion Practice in Renal Transplants Recipients  77

Unlike the B lymphocyte mediated hyperacute rejection,
this reaction is mediated through alloreactive T cells.2
Activated cytotoxic T cells infiltrate the graft and trigger an
immune response. They can induce graft injury by inducing
apoptosis and by secreting cytotoxic proteins (perforin and
granzyme B). Pre-transplant assessment for the presence or
absence of alloantibodies and T cell activities to HLA antigens
reduces the risk of acute rejection.
Humoral Rejection
Outside of the hyperacute rejection state, humoral rejection
can still occur although less frequently than cell mediated
acute rejection.3 Humoral rejection is characterized by B
lymphocytes injuring the allograft through immunoglobulin
and complement activities.1
Chronic Rejection
The definition of chronic rejection is ambiguous, and is
sometimes recognized as chronic allograft nephropathy or
any immunological responses that results in slow loss of graft
function with histopathological processes: tubular atrophy,
interstitial fibrosis, and fibrous intimal thickening of arteries.4
Evolution of Transfusion in Renal
Transplantation
High-volume therapeutic use of blood transfusion was
originally used in attempts to maintain ESRD patients who
were anemic with red cell mass ranges of 20–25%. Due
to concerns with transfusion-induced infections such as
hepatitis and the production of anti-HLA antibodies resulting
from the exposure to blood products, efforts were made
in the 1970s to avoid the use of blood transfusions in renal
transplant recipients.3
During the pre-cyclosporin era, studies suggested that
non-transfused renal graft recipients were at higher risk for
graft rejection as compared to those transfused recipients5,6
(Table 1).
Subsequently, many studies attempted to define the
optimal dose and timing for the transfusion effect. With
the introduction of cyclosporin in the early 1980s, leading
to improved renal graft and patient survival, the beneficial
role of blood transfusions and HLA matching was again
questioned.18 Meanwhile, some preliminary trials had shown
the use of per-protocol transfusion: matched pre-transplant
blood transfusion (motifs) or donor-specific transfusion
(DST) to be beneficial. The evidence supporting the
effects (positive, negative, neutral) of pre-transplant blood
transfusion, regardless of therapeutic or protocol transfusion,
in renal transplantation is still not well-established. It is
unclear whether the benefits, if any, of pre-transplant blood
transfusion may be due to the modulation of immune
response in which tolerance is induced.
There are two keys questions that need to be answered for
understanding of effect of transfusion on renal transplant.
Question 1. Do red blood cell transfusions prior to renal
transplant impact allograft rejection/survival and what is
the magnitude of that effect relative to other factors (e.g.
pregnancy, prior transplantation)?
In summary, prior transplant may induce increase in
rejection and worsening graft survival or no significant
changes may occur but benefits are unlikely to result from
these outcomes. Transfusions may be related to decreasing
rejection and benefiting graft survival or no significant
changes may occur but it is unlikely that these outcomes
would worsen as a result of transfusion (Table 1). Prior
pregnancy had very scant data and could not be well assessed
(Table 2).

Table 1: Impact of transfusion on rejection, graft and patient survival

Rejection Graft survival Patient survival References
Transfusion Significant beneficial Significant beneficial to no Beneficial to neutral effect 7-14
versus to no significant effect on 1-year on 1-year and maximum
No transfusion significant effect on and maximum duration duration patient survival
rejection outcomes graft survival

Table 2: Impact of prior transplant and pregnancy related to transfusion on rejection graft and patient survival
Study , year Anaylsis type Outcome Multivariate results Significant effect
Impact of previous transplant
Reed A, 1991 Poisson Rejection episodes NR, NA
N=12715 P=0.40
Sanfilippo F, 1986 Cox Irreversible graft RR 1.409, Transplant worsens
N=381116 rejection P=0.0002 rejection
Sanfilippo F, 1986 Cox Irreversible graft RR 1.884, Transplant worsens
N=381116 rejection P=0.0006 rejection
Impact of pregnancy
Reed A, 1991 Poisson Rejection episodes NR, Pregnancy benefits
N=12715 P=0.017 rejection
78  Section 1  Clinical Hemotherapy
Question 2. Is any such impact of red blood cell transfusions
on renal transplant outcomes altered by variables such as:
• The number of transfusions, the number of units of blood,
and/or the number of donors (Table 3).
• The use of leukocyte depleted blood (Table 4).
HLA and ABO Sensitization and
Desensitization in Renal Transplantation
Despite a marked increase in the number of living donor
allografts (LDA) performed annually, many potential
recipients with otherwise suitable donors are relegated to
the ever expanding deceased donor waiting list secondary
to either: Preformed human leukocyte antigen (HLA) anti­
bodies. These are acquired via pregnancy, transfusion, or
prior transplant or ABO blood group incompatibility (ABOi).
If not adequately removed, the presence of such antibodies
is likely to result in severe antibody mediated rejection (AMR)
and early graft loss.
Patients with a willing but HLA- or ABO- incompatible
donor may wait for a deceased donor organ or receive a living
donor organ through desensitization, kidney paired donation
(donor swap) or a combination of both. The choice between
desensitization and kidney paired donation for an individual
recipient depends upon the likelihood of finding a suitable
match through kidney paired donation and the chances of
successful desensitization. The increased risk of hyperacute
antibody mediated rejection (AMR) and subsequent allograft
loss when transplanting against either preformed HLA
antibodies or ABOi has engendered a general avoidance
of this practice and started using various desensitization
protocols designed to reduce the amount of pre-existing
antibody to a level that allows for successful engraftment
leading to successful transplantation across HLA and/or ABO
barriers.
ABO Desensitization
Major incompatibility refers to the presence of natural
antibodies in the recipient against the donor’s A or/
Table 3: Impact of number of transfusion on rejection, graft and patient survival
Rejection Graft survival
Number/Units of Beneficial to neutral Beneficial to no significant
transfusion, effect on rejection effect on 1-year and
Number of donors outcomes maximum
duration graft survival
Table 4: Impact of leukodepletion on rejection, graft and patient survival
Rejection Graft survival
Leukocyte-depleted NA Beneficial effect on 1-year
versus therapeutic and maximum duration
transfusion graft survival
and B blood group antigen. These antibodies may cause
hyperacute/acute humoral rejection of the organ due to
endothelial damage because A and B antigens are expressed
on the vascular endothelium. ABOi exists in approximately
35% donor-recipient pairs by virtue of ABO blood group
distribution in general population. The A2 blood group
has reduced expression of A antigen on their RBCs and
endothelium and, therefore, group A2 donors are preferred
over group A1 donor for group O or B recipients in kidney
transplantation with very low-risk for graft rejection. Minor
incompatibility occurs where the organ donor has naturally
occurring ABO antibodies against the recipient.
Current Management/Treatment
Most published reports on ABOi solid organ transplantations
suggest removal of anti-A or anti-B antibodies in conjunc­
tion with immunosuppressive treatment with drugs such as
tacrolimus and Mycophenolate mofetil (MMF); and mono­
clonal antibodies like Daclizumab and Rituximab. Rituximab
is effective in B cell ablation but does not affect plasma cells.
Other immunotherapy modalities including intravenous
immunoglobulins (IVIG) and antithymocyte globulins (ATG)
have important roles in the transplant process.
Rationale for Therapeutic Apheresis
There are no controlled clinical trials using TPE in ABOi solid
organ transplantations. Due to past experiences of hyperacute
and acute rejection of ABOi solid organs, TPE has been used as
an adjunct therapy to reduce anti-A or anti-B antibody titres in
the peri-transplant period, thus preventing hyperacute/acute
rejection and improving graft survival. TPE has been included
in preparatory regimens for ABOi solid organ transplantation
as an adjunct with different immunosuppression therapies
and IVIG. In ABOi kidney transplantation, TPE is part of a
preconditioning protocol to lower antibody titre to <8/<4
prior to the transplant procedure. Apart from TPE, specific A
or B antigen immuno-adsorption columns have been used in
Europe to selectively remove anti-A or anti-B antibodies.
Patient survival References
Neutral effect on 1-year 16-18
and maximum duration patient
survival
Patient survival References
Neutral effect on 1-year and 19
maximum
duration graft survival
 Chapter 22  Transfusion Practice in Renal Transplants Recipients  79
Duration and Discontinuation/
Number of Procedures
The goal should be to reduce the antibody titre (IgM and IgG)
to ≤ 8/4 in renal transplantation. This titre can be achieved
usually in 2–5 days, depending upon the baseline titres. The
antibody titres may increase 3–7 days after transplantation;
therefore, daily antibody titre for the first 2 weeks post-
transplantation is necessary. During the following 2 weeks,
antibody titre measurement every second day helps to
prevent immunologic graft events. If the antibody titre is
high (>32) with or without humoral rejection, TPE should be
performed again in the post-transplantation period. In renal
transplantation, usually three more TPEs are performed
postoperatively (every second or third day followed by IVIG).
If the antibody titre can be maintained at <8 in post-transplant
first week and 16 in second week, the risk of humoral rejection
is decreased.
HLA Desensitization and AMR
Patients with an elevated HLA antibody screen have difficulty
finding an HLA compatible donor and remain on the
transplantation list significantly longer than unsensitized
patients. The goal of desensitization protocols is to allow
these individuals to be transplanted using a donor kidney
that would otherwise not be usable due to the high likelihood
of graft loss.
Allograft rejection has traditionally focused on T cell
mediated process causing cellular rejection. Recently a clear
histological diagnosis of AMR can be made based on the
Sixth Banff Conference on Allograft pathology in 2007. The
diagnosis is based on:
• Documentation of donor specific antibody (DSA).
• Histologic evidence of acute tissue injury, such as acute
tubular injury, neutrophils in peritubular capillaries and/
or glomeruli, and/or capillary thrombosis, or intimal arte­
ritis/fibrinoid necrosis/intramural or transmural inflam­
mation in arteries.
• C4d in peritubular capillaries or immunoglobulin and
complement in arterial fibrinoid necrotic areas by
immunohistology.
AMR affects fewer than 10% of renal allografts. Recipients
at higher risk include those with previous transplant and high
panel-reactive antibodies.
Current Management/Treatment
New immunosuppressive drugs are continually being
developed to prevent and treat acute renal allograft rejection,
and to decrease antibody titres. All transplant recipients are
placed on immunosuppressive therapy but individuals with
a high likelihood of acute rejection, including those with
HLA antibodies and recipients of cadaveric organs, receive
more intense regimens. The optimal regimen has yet to be
defined but include the use of cyclosporine, tacrolimus,
mycophenolate mofetil, azathioprine, and anti-thymocyte
globulin.
Treatment of AMR includes increasing immunosuppres­
sive medications as well as rituximab, TPE, IVIG or splenec­
tomy. In severe cases or cases refractory to TPE+IVIG,
splenectomy and rituximab have been used. Clinical trials
have demonstrated improved graft survival with TPE/IVIG
versus TPE alone or IVIG alone, and TPE + rituximab versus
TPE alone.
Rationale for Therapeutic Apheresis
In antibody-mediated rejection DSA are generated after
transplantation. These antibodies can be removed with
plasma exchange, double filtration plasmapheresis and
immunoadsorption. Therapeutic apheresis is always in
combination with other immunosuppressive drugs, such as
antithymocyte-globulin, glucocorticosteroids, rituximab, and
intravenous immunoglobulin.
TPE can also be used prior to transplant to remove HLA
antibodies. TPE (some series have used double filtration
plasmapheresis and one small series used Prosorba column)
is used in combination with immunosuppressive drugs
pre-transplant until cross-match is negative. TPE is usually
continued postoperatively also and re-initiated in cases
where AMR occurs. The ability to obtain a negative cross-
match depends on the DSA titre. Using approximately 5 TPE
pre-operatively, will allow the titre of < 32 to become negative.
Duration and Discontinuation/
Number of Procedures
For AMR, some protocols use a set number of procedures,
usually 5 or 6, daily or every alternate day. Other protocols
guide number of treatments based on improvement in renal
function and decrease in DSA titres. It is also undecided if low
dose IVIG (100 mg/kg) should be used after every procedure
or at the end of the series or not at all. For desensitization
protocols, TPE is performed daily or every other day per
protocol until cross-match becomes negative. TPE is also
performed postoperatively for a minimum of 3 procedures.
References
1. Gabardi S, Olyaei AJ. Solid organ transplantation, chapter 55.
In, Chisolm-Burns MA, Ed. Pharmacotherapy Principles and
Practice, 2nd edn. McGraw-Hill, NY, NY. 2010. pp. 939-64.
2. The Organ Procurement and Transplant Network (OPTN).
Available at: http://optn.transplant.hrsa.gov/latestdata/
viewdatareports.asp. Accessed: 2010.
3. Opelz G, Sengar DP, Mickey MR, et al. Effect of blood
transfusions on subsequent kidney transplants. Transplant
Proc. 1973;5:253-9.
4. Halloran PF. Call for revolution: a new approach to describing
allograft deterioration. Am J Transplant. 2002;2:195-200.
80  Section 1  Clinical Hemotherapy
5. Opelz G, Terasaki PI. Improvement of kidney-graft survival
with increased numbers of blood transfusions. N Engl J Med.
1978;299:799-803.
6. Fuller TC, Delmonico FL, Cosimi B, et al. Impact of blood
transfusion on renal transplantation. Ann Surg. 1978;187:211-8.
PMID: 343736.
7. Marti HP, Henschkowski J, Laux G, et al. Effect of donor-
specific transfusions on the outcome of renal allografts in the
cyclosporine era. Transpl Int. 2006;19:19-26.
8. Hiesse C, Busson M, Buisson C, et al. Multicenter trial of one
HLA-DR-matched or mismatched blood transfusion prior to
cadaveric renal transplantation. Kidney Int. 2001;60:341-9.
9. Alexander JW, Light JA, Donaldson LA, et al. Evaluation of pre-
and post-transplant donor-specific transfusion/cyclosporine A
in non-HLA identical living donor kidney transplant recipients.
Cooperative Clinical Trials in Transplantation Research Group.
Transplantation. 1999;68:1117-24.
10. Opelz G, Vanrenterghem Y, Kirste G, et al. Prospective
evaluation of pretransplant blood transfusions in cadaver
kidney recipients. Transplantation. 1997;63:964-7.
11. Sharma RK, Rai PK, Kumar A, et al. Role of preoperative donor-
specific transfusion and cyclosporine in haplo-identical living
related renal transplant recipients. Nephron. 1997;75:20-4.
12. Aalten J, Bemelman FJ, van den Berg-Loonen EM, et al.
Pre-kidney-transplant blood transfusions do not improve
transplantation outcome: a Dutch national study. Nephrol Dial
Transplant. 2009;24:2559-66.
13. Albrechtsen D, Flatmark A, Brynger H, et al. Impact of blood
transfusions and HLA matching on national kidney transplant
programs: the first Swedish-Norwegian Study of cyclosporine.
Transplant Proc. 1988;20:257-60.
14. Barbari A, Stephan A, Masri MA, et al. Donor specific
transfusion in kidney transplantation: effect of different
immunosuppressive protocols on graft outcome. Transplant
Proc. 2001;33:2787-8.
15. Reed A, Pirsch J, Armbrust MJ, et al. Multivariate analysis
of donor-specific versus random transfusion protocols in
haploidentical living related transplants. Transplantation.
1991;51:382-4.
16. Sanfilippo F, Vaughn WK, LeFor WM, et al. Multivariate
analysis of risk factors in cadaver donor kidney transplantation.
Transplantation. 1986;42:28-34.
17. Corry RJ, West JC, Hunsicker LG, et al. Effect of timing of
administration and quantity of blood transfusion on cadaver
renal transplant survival. Transplantation. 1980;30:425-8.
18. Melzer JS, Husing RM, Feduska NJ, et al. The beneficial effect
of pretransplant blood transfusions in cyclosporine-treated
cadaver renal allograft recipients. Transplantation. 1987;43:
61-4.
19. Nube MJ, Persijn GG, van Es A, et al. Beneficial effect of HLA-A
and B matched pretransplant blood transfusions on primary
cadaveric kidney graft survival. Transplantation. 1983;35:556-
61.
Incompatible Blood Type
Kidney Transplantation
Varun Sharma, Priyadarshi Ranjan
Human leukocyte antigen (HLA) sensitization and ABO
incompatibility continue to pose a significant barrier to
expansion of living donation. In fact, either anti-blood
or anti-donor HLA antibodies result in the occurrence of
hyper acute rejection and graft loss. Reducing this early
rejection risk by planned desensitization protocols has
clearly improved the outcome of ABO-incompatible (ABOi)
kidney transplantation. B-cell depletive therapy has replaced
splenectomy, overcoming the disadvantages of early graft
loss and early rejections episodes and, consequently,
improved the latter. Plasma exchange techniques have
considerably reduced antibody titers, allowing better results.
Thus, newer immunosuppressive protocols reduced long-
term graft survival. Therefore, ABOi kidney transplantation
can be more broadly practiced, especially to expand the pool
donor and to reduce the waiting time for transplantation.
Kidney transplantation (KT) is known as a standard therapy
for patients with end-stage kidney disease (ESKD) and has
been adopted widely in the world. However, the living and
deceased kidney donor pool does not resolve the shortage of
transplantable organs. Different ways have been proposed to
increase the donor pool and ABO incompatible KT (ABOi-
KT) represents a valid source of organs to decrease the
donor waiting list. ABOi-KT requires extra strategies and
suffers extra risks across ABO blood type barrier compared
to ABO compatible KT (ABOc-KT). ABOi-KT was previously
considered to be contraindicated for many years. Presently,
ABOi-KT has been accepted as a valid alternative therapy for
ESKD and the outcome of ABOi-KT has become equivalent
to ABOc-KT in adult and pediatric recipients. When a patient
with ESKD requires KT and an acceptable living donor is ABO
incompatible with the recipient, the patient can currently
chose one of three options: (1) stay on the waiting list for
deceased donor KT; (2) have paired kidney donor exchange
(PKDE); or (3) undergo ABOi-KT.
Paired kidney donor exchange (PKDE) is an innovative
method whereby 2 or more incom­patible donor-recipient
pairs exchange donors to create 2 or more compatible pairs.
It is a very reasonable idea for human leukocyte antigen
(HLA) sensitized and/or ABO incompatible patients. This
23
primary idea was reported first by Rapaport in 1980s. There
are currently several variations of exchange such as three-
way, four-way and domino paired donation. PKDE provides a
recipient with an incompatible donor the chance to receive a
compatible kidney, which is available by expanding the donor
source and reducing the waiting time for deceased donor KT.
Advantages of PKDE are low immunological risk, avoidance
of intensified immunosuppression due to desensitization,
and cost effectiveness.
Alexandre et al. demonstrated the ABOi-KT strategy using
plasmapheresis and splenectomy to break the ABO barrier.
This has been used as a desensitization strategy for ABOi-KT
for 20 years. ABOi-KT has become common in Japan due to
the lack of deceased donors, and ABOi-KT has accounted
for approximately 30% of all living-donor KT in that country.
On the contrary, a tiny proportion, only 738 cases (0.94%)
of ABOi-KT were performed between 1995 and 2010 in the
United States, but this number is increasing annually. The
same trend continues in the United Kingdom: over the last
decade, there has been an increase of ABOi-KT from less
than 10 per year to 100 per year representing 1.0% of living
donor transplants performed. This increase is possibly due
to the fact that protocols have been simplified over the years
from complex surgical and pharmacological processes that
variably may have involved splenectomy, rituximab (RIT),
plasmapheresis and antibodies titration.
ABO ANTIGENS AND ANTIBODIES
The concept of blood groups A, B and O was established by
Nobel laureate Karl Landsteiner in the early 1900s. These are
polysaccharide antigens which are found in red cell, platelets,
and other tissues such as endothelium. The antibodies to
blood group antigen are isohemagglutinins and can be of
either immunoglobulin M (IgM) or immunoglobulin G (IgG)
type antibodies. However, in the context of transplantation it
is IgG that is functionally significant. Blood type A develops
anti-B antibody, and blood type B has anti-A antibody. Blood
type AB with A and B antigen has both antibodies, while
blood type O with both antibodies does not have any antigen.
82  Section 1  Clinical Hemotherapy
Blood type incompatibility means the exposure of A or B
antigen to a person who has antibodies against these antigens.
Therefore, these antigen expressions of an organ have been
obstacles for ABOi-KT.
All blood type recipients accept a blood type O donor as a
universal donor, and a blood type AB accepts all blood type
donors as a universal recipient. Blood group type A, however,
carries A1 or A2. The expression of A2 antigen is weaker than
that of A1 antigen. The A2 subtype constitutes approximately
20% of blood type A in white races, while it is only 0.15% in
Japanese population. A2 kidney may be less likely to suffer
antibody rejection in the presence of anti-A antibody. In
fact, non-A recipients receiving kidneys from A2 donors, can
universally and safely accept the transplantation without
preconditioning at times of KT.
HISTORY
Splenectomy, Rituximab and
No B-cell Depletion
The first successful report of ABOi-KT is dated back to 1987
when authors achieved long-term allograft survival in a
series of 23 patients. Plasmapheresis and splenectomy
were performed to reduce anti-blood type A or B (anti-A/B)
antibody and to minimize the risk of hyperacute humoral
rejection. Most of the modern desensitization protocols of
ABOi-KT have been derived from this procedure and have
since evolved. Their work was further greatly expanded in
Japan due to the shortage of deceased donors with successful
outcomes in ABOi-KT.
Now-a-days, splenectomy has been totally abandoned
and the various desensitization protocols in use are combi­
nations of antibody removal by plasmapheresis or immuno­
adsorption (IA), intravenous immunoglobulin (IVIG) to
neutralize preformed antibodies, B lymphocyte depletion
by anti-CD20 monoclonal antibody (RIT) and standard
triple immunosuppression (calcineurin inhibitor, CNI;
mycophenolate mofetil, MMF; and steroid). Recently, some
authors reported successful outcomes of ABOi-KT without
RIT and splenectomy.
ABOi-KT PREOPERATIVE MANAGEMENT
Current strategies of ABOi-KT compose three common
principles: (1) antibody measurement; (2) B-Cell depletion;
and (3) antibody depletion.
Antibody Measurement
Assessment of anti-A/B antibody titer is crucial in ABOi-KT.
It guides the effectiveness of operative preconditioning and
determines the period to permit transplantation. In addition,
posttransplant monitoring helps early detection of antibody-
mediated rejection (AMR) by antibody rebound.
There are various measurement methods of anti-A/B titer,
the most common used are tube technique, gel technique
and flow cytometry. Although each center uses their familiar
technique, there is a discrepancy of measured titer level.
Kobayashi et al. surveyed the differences of anti-A/B titers from
the same blood samples, which were measured by tube test
in 29 Japanese centers. It was revealed that inter-institutional
differences were 1:8 to 1:32 in IgM and 1:16 to 1:256 in
IgG, because of low reproducibility by visual observation.
Therefore, they concluded standardized measurement
should be necessary. Kumlien et al. analyzed the same blood
samples in three centers. They also pointed out an inter-center
variation of titer level using tube technique and suggested
that gel technique is more reproducible than tube technique.
Flow cytometry showed excellent reproducible compared
with other techniques and would be suitable for the accurate
measurement. However, this technique is not available in all
centers due to the expensive equipment required.
High preoperative anti-A/B IgG titers are associated with
poor long-term allograft survival in ABOi-KT. Gloor et al.
showed preoperative high anti-A/B IgG titers are a predictor
for AMR, and the rapid increasing of titers is also associated
with AMR and graft loss. In addition, Tobian et al. also
demonstrated that AMR was also associated with high titer
at 1–2 wk posttransplant. Chung et al. described there was
no statistically significant difference between high- (> 1:256)
and low-titer (< 1:128) at the baseline in allograft function at
6 months after transplantation. Therefore, appropriate
monitoring of anti-A/B titer is essential before and after ABOi-
KT. Although anti-A/B antibody titer has to be measured
during the early period after ABOi-KT due to the risk of AMR,
but how long the monitoring should be continued remains
unclear. Preoperative titer should be low in ABOi-KT, but the
acceptable titer of anti-A/B antibody at the time of transplant
has varied between 1:4 and 1:32 in line with the protocol of
individual centers. After the ABO incompatible transplant
necessitating initiation of antibody-depletion procedures,
the level of anti-ABO antibody titer must be monitored to
detect rebound in the serum antibody production.
B-cell Depletion
Splenectomy: Splenectomy was considered a prerequisite
for desensitization protocol in ABOi-KT after Alexandre
et al. reported that it reduced the risk of AMR. The principle
of splenectomy was based on the concept that spleen is
reservoir of antibody producing B-cells and antibody-
producing plasma cells in the body. However, the efficacy
of splenectomy in ABOi-KT is debatable, because severe
AMR sometimes still occurs after splenectomy. The effect of
splenectomy on the immune system is permanent. Following
splenectomy the patients are at risk for the development
of life-threatening sepsis, especially from encapsulated
bacteria and they require life-long antibiotic prophylaxis.
Splenectomy can lead to surgical complications such as
 Chapter 23  Incompatible Blood Type Kidney Transplantation  83
hemorrhage, pancreatic injury, pancreatic leakage, and
portal vein thrombosis.
A comparative analysis of splenectomized recipients
compared with RIT treated but without splenectomy, showed
no statistically significant difference in the anti-A/B titer of KT
and liver transplantation. It was concluded that splenectomy
was not an essential prerequisite treatment in ABOi-KT.
Although splenectomy has been replaced with RIT, Locke
et al. reported that splenectomy could be useful as salvage
treatment for severe AMR secondary to anti-HLA antibody.
Current consensus states that splenectomy is not necessary
for the induction of ABOi-KT.
Rituximab: Splenectomy has been largely replaced by RIT
in ABOi-KT protocols to remove B-cell. RIT is an anti-CD20
monoclonal antibody, which binds to CD20 on immature
and mature B-cell resulting in depletion of B-cell. RIT was
originally developed for the treatment of non-Hodgkin’s
lymphoma. RIT has been used extensively in the treatment
of patients with autoimmune diseases and KT besides
hematological malignancies. Adverse events related to B-cell
depletion by RIT include fever, chill, headache, and nausea,
whilst serious cardiovascular and pulmonary events are rare.
In the field of KT, RIT has been used as a part of desensi­
tization protocols in ABO- and HLA-incompatible KT, treatment
of AMR, post-transplant lymphoproliferative disorder, and
recurrent nephrotic syndrome. In the first experience of RIT use
in ABOi-KT recipients, Sawada et al. tried RIT, splenectomy,
and double-filtration plasmapheresis (DFPP) for A1 to O ABOi-
KT with persistent high anti-A antibody titer. The dosage of
RIT was 375 mg/m2 per week for 4 wk pretransplant and there
was no rebound of the titer after transplantation. Tydén et al.
succeeded with 4 ABO incompatible recipients using RIT and
antigen-specific IA (IAs) with standard immunosuppression,
without splenec­tomy. In their protocol, RIT (375 mg/m2) was
administered once 10 d prior to transplant which was enough
to deplete peripheral B-cell. Moreover, its effect was long-
active for at least 12 months without any serious side effects.
After these successful reports were published, RIT has replaced
splenectomy in desensitization protocol. Recently, some have
tried low dose of RIT or even omitting it in ABOi-KT protocol
to avoid over-immunosuppression without compromising
excellent outcomes.
Twenty-seven recipients who were diagnosed with steroid-
resistant cell-mediated rejection or AMR received a single
dose of RIT (375 mg/m2) as a salvage treatment twenty-four
(88.9%) among these demonstrated improved renal function.
Serum creatinine decreased from a mean of 5.6 mg/dL before
the treatment to a mean of 0.95 mg/dL after the treatment.
RIT is useful not only in AMR, but also in chronic antibody-
mediated rejection (CAMR) prevention. Kohei et al. observed
that ABOi-KT with RIT had a statistically significant lower
rate of CAMR at 2 years posttransplant than living ABOc-KT
(3.5% vs 28.9%). However, this beneficial effect of RIT needs
independent verification.
Antibody Depletion
The antibody depletion treatments are the basis of ABOi-
KT. In order to eliminate existing anti-A/B antibody, plasma
exchange (PE), DFPP and IA are available. They differ in their
mechanisms of action, specificity, efficiency and cost.
In PE, plasma is removed and replaced by human
albumin, colloid solutions, and/or fresh frozen plasma
(FFP). It has been widely used around the world for antibody
removal in ABOi-KT. This method is simple, but it has several
disadvantages compared with more specific techniques.
Because of nonselective apheresis, PE removes not only
anti-A/B antibody, but also coagulation factors and anti-
viral/-bacterial immunoglobulin. Consequently, the risk of
bleeding and infection is increased. FFP is generally needed
for the last session before KT to prevent these complications.
Other complications were reported by Tobian et al. In all PE
sessions (n = 512), the total rate of complications was 15.4%.
The most common complication was hypocalcemia (6.8%),
followed by urticaria or pruritus (4.3%), hypotension (2.9%)
and nausea or vomiting (1.2%).
DFPP is designed to remove selectively the immunoglobulin
from plasma and requires less substitution fluid compared to
PE. When plasma separated by a first filter is passed through
a second filter, IgG and IgM are filtered out and discarded. By
single DFPP, 70% of IgM and 60% of IgG were removed and a
one-fold titer reduction of anti-A/B antibody was observed.
This technique also avoids the loss of coagulation factors and
albumin unlike PE. However, significant amounts of albumin
are lost by DFPP, and almost always albumin is needed as
the replacement fluid. DFPP is also removes variable amount
of fibrinogen, and its measurement is necessary to avoid
bleeding complication.
IA can be A/B antigen IAs or A/B nonantigen IAns (non-
specific/semi-selective immunoadsorption) respectively if it
removes only a specific antibody such as anti-A/B antibody
or removes nonantigen-specific immunoglobulin. Between
the two techniques IAs is most utilized method in ABO
incompatible setting. On the other hand, IAns is suitable
for the elimination of HLA antigens and it is most used in
HLA incompatible/ABOi KT recipients. In IAs, the plasma
is processed through an ABO immunoadsorbent column,
which is coated with either blood type A or B antigens and
allow selective removal of anti-A or B antibody, and the
processed plasma is re-infused into the patient. Volume
replacement is not necessary. IAs is selective and free from
side effects of PE and DFPP. Single IAs reduces 2- to 4-fold
titer between pre- and post-IAs, and at least four preoperative
IAs are usually needed to obtain an acceptable titer at the
expense of increased cost compared to PE and DFPP. IAs is
generally safer and more effective, and therefore normally
preferred. However, ultimate choice depends on each centre’s
decision, based on the availability of infrastructure and skills
of staff.
84  Section 1  Clinical Hemotherapy
USE OF IVIG
IVIG’s recognized immunomodulatory properties have been
employed for the treatment of autoimmune diseases. IVIG
is believed to act through various mechanisms: (1) comple­
ments down-regulation; (2) interactions with the Fc receptors;
(3) inhibit of B/T-cell proliferation; (4) inhibit of CD8 T-cell
cytotoxicity; and (5) increased apoptosis of B-cell. Mild and
early adverse effects of IVIG include headache, chills, nausea,
fatigue, myalgia, arthralgia, chest pain, back pain, and
elevated blood pressure. However, rare but serious delayed
adverse effects include renal toxicity, thromboembolic events
(cerebrovascular accident and deep venous thrombosis),
neurological toxicity (aseptic meningitis), hematological
toxicity (neutropenia), and dermatological toxicity. The
administration of high dose IVIG can cause hemolysis by anti-
A/B antibody within the IVIG. In ABOi-KT, it is preferable if
possible to use IVIG with low anti-A/B titer in order to avoid
not only hemolysis but also AMR after transplantation due to
anti-A/B titer elevation.
There is no uniformity in the dose IVIG used in the
desensitization protocols of ABOi-KT. IVIG is usually adminis­
tered after plasmapheresis, to reconstitute the natural levels
of IgG. In the absence of control data, the use of IVIG in ABOi-
KT can best be described as empirical.
ACCOMMODATION
Without adequate anti-A/B antibody reduction and desensiti­
zation before KT, an incidence of AMR and irreversible
damage cannot be avoided. Successful ABOi-KT requires the
reduction of anti-A/B antibody titers against ABO antigens on
the graft at the time of KT. However, anti-A/B antibody titer
returns to the baseline level within almost 1 wk after KT, even
if optimal desensitization is performed. Therefore, intense
monitoring is necessary during critical first two weeks after
ABOi-KT. Paradoxically, a phenomenon of accommodation
is acquired in this term.
Accommodation is defined as a phenomenon whereby
graft rejection is avoided despite reemergence of incompatible
antibody. The mechanism was originally discovered in
the field of xenotransplantation, whereby endothelial cell
posttransplant humoral injury was avoided, possibly due to
changes of antibody specificity, avidity, affinity and alteration
of the antigen structure. This phenomenon is allegedly
responsible for normal graft function and structure despite
reemergence of anti-A/B antibody against incompatible
A or B antigen in the graft; However, it is fair to accept that
mechanism as well as the very existence of accommodation
remains speculative.
CURRENT PROTOCOL OF ABOi-KT
In ABOi-KT, intensified immunosuppressive protocol usually
starts before KT in order to deplete anti-A/B antibody.
Many centers have modified original successful protocol of
ABOi-KT.
RIT has been adopted in the place of splenectomy by
majorities of centers. However, the timing and dose of
RIT administrated remains variable. RIT or splenectomy-
free protocols have successfully, used low dose IVIG after
plasmapheresis. The basis of the North Europe protocol is IAs
followed by high dose IVIG. However, postoperative IAs is not
performed routinely and its use is determined by antibody
titers. Maintenance immunosuppressive agents are mostly
triple agents which are CNI, MMF and steroid. Tacrolimus is
the CNI of choice in these ABOi-KT protocols. MMF was taken
7–14 d pretransplant in order to inhibit antibody production.
Some centers use a protocol without daclizumab, basiliximab
or antithymocyte globulin, and report excellent outcomes.
Thus it is controversial whether these clonal antibodies
should be introduced in ABOi-KT or not. All protocols of
ABOi-KT have resulted in satisfactory outcome in the absence
of randomized control trials. It is impossible to select an ideal
protocol fit for all purpose.
MINIMIZE IMMUNOSUPPRESSION
Efforts have been made to minimize immuno­sup­pression
in order to reduce the long-term risk of over-immuno­
suppression. The long-term effect of steroid use remains
unclear in ABOi-KT. Oettl et al. described 11 ABOi-KT
recipients with late steroid withdrawal. Six recipients showed
biopsy-proven acute rejection during or soon after steroid
cessation. However, Galliford et al. tried early steroid sparing
protocol in 10 recipients. Prednisolone was maintained at 1
mg/kg until 3 d posttransplant. It was reduced to 0.5 mg/kg at 4
d posttransplant, and discontinued after 1 wk posttransplant.
In this study, patient and graft survival were 100% at 1 year
posttransplant but 3 patients experienced acute rejection
within 1 mo after transplantation.
HISTOLOGICAL FINDINGS IN ABOi-KT
In ABOi-KT, acute AMR by anti-A/B antibody is a well-
recognized cause of early graft loss. Diagnosis of acute AMR
needs C4d staining in the peritubular capillary (PTC) and
the presence of anti-donor antibodies. Morphologic changes
include acute tubular necrosis, capillary and/or glomerular
inflammation, and transmural arteritis and/or arterial
fibrinoid change. C4d staining is the hallmark of humoral
induced complement activation and like ABOc-KT was
thought to be a useful indicator of AMR even in the setting
of ABOi-KT. However, C4d deposition without AMR was seen
in 85.7% of ABOi-KT at 3 mo posttransplant. Setoguchi et al.
analyzed protocol biopsies of ABOc-KT and ABOi-KT. C4d
expression of PTC was detected in 94% of ABOi-KT, whereas
in only 11% of ABOc-KT. In protocol biopsies during stable
allograft function, 80% of ABO incompatible grafts showed as
C4d positive, while 74% of HLA incompatible grafts were C4d
 Chapter 23  Incompatible Blood Type Kidney Transplantation  85
negative.These histological studies indicate that the detection
of C4d alone in ABO incompatible graft does not indicate
AMR and support a concept of accommodation in ABOi-KT.
Therefore, AMR after ABOi-KT can only be diagnosed on the
basis of morphological evidence, serological evidence and
the clinical course.
Morphologically transplant glomerulopathy (TG) at 1 year
after transplantation was reported as an indicator of poor
outcome. ABOi-KT had more severe TG than ABOc-KT without
HLA antibody at 1 year posttransplant. However, there were
no differences in interstitial fibrosis, tubular atrophy, chronic
vasculopathy and allograft function between both groups.
In the absence of prior AMR, histological change at 1 year
posttransplant was mild irrespective of ABO compatibility.
Moreover, prior AMR in ABOi-KT was associated with TG
and interstitial fibrosis and not to arteriolar hyalinosis and
chronic vasculopathy. Consequently, ABO incompatible
grafts with TG and/or interstitial fibrosis had lower GFR at
1 year after transplantation than those with normal histology.
INCIDENCE OF ACUTE CELLULAR AND
ANTIBODY MEDIATED REJECTION IN
ABOi-KT
As previously described, the outcome of graft survival in
ABOi-KT has been similar to ABOc-KT. However, there is an
increased risk of AMR in ABOi-KT due to anti-A/B antibody.
Protocol biopsies at 3 months post-transplant in ABOi-KT
had a significantly higher incidence of AMR compared to
ABOc-KT (17.9% vs 1.1%). However, there was no significant
difference in the rate of acute cellular rejection between ABOi-
KT and ABOc-KT (48.4% vs 35.7%) In the acute lesion score
based on Banff classification, t2-3 and g2-3 following ABOi-KT
was higher than that of ABOc-KT (t2-3: 42.9% vs 19.4%, g2-3:
28.6% vs 6.5%). Gloor et al. described in the study of protocol
biopsies at 1 year posttransplant that there was a significant
difference in the incidence of acute rejection between ABOi-
KT and ABOc-KT without HLA antibody (50% vs 13.6%). Acute
rejection in ABOi-KT was mainly AMR (73.3%) as compared
to ABOc-KT without HLA antibody (12.5%). Setoguchi et al.
also compared the histologic findings of protocol biopsies in
48 ABO incompatible and 133 compatible grafts. There was no
difference in clinical and subclinical rejection between ABO
incompatible and compatible grafts (clinical: 37.5% vs 25.6%,
subclinical: 10.4% vs 15%). However, ABO incompatible grafts
had a high incidence of AMR compared to ABO compatible
grafts (27% vs 5.3%). Interestingly, rejection was detected in
only 15.0% at 1 mo in ABOi-KT compared to 34.7% in ABOc-
KT, but in 30.0% at 6–12 mo compared to 10.5%. Wilpert et al.
demonstrated that the rejection rates in ABOi-KT were similar
to that in ABOc-KT. Acute cellular rejection was detected in
23.2% of ABOi-KT and in 22.5% of ABOc-KT. Acute AMR was
shown in 4.7% of ABOi-KT, which was similar to ABOc-KT
(5.0%).
ADVERSE EFFECT OF ABOi-KT
Infection
The improvement in ABOi-KT graft survival rate has come
at the expense of increased posttransplant infection. The
infection rate in ABOi-KT is significantly higher than in
ABOc-KT (60% vs 29.8%). The rates of infection including
cytomegalovirus (CMV), herpes simplex virus, varicella zoster
virus and BK virus (BKV) in ABOi-KT were also significantly
higher than in ABOc-KT. The most common viral infection
was BKV in 25% of ABOi-KT compared to only 8.5% of ABOc-
KT. However, the incidences of rejection, graft survival rate
and function of ABOi-KT patients were compatible with these
of ABOc-KT patients. On the contrary, Genberg et al. showed
that there was no statistical difference in overall infection
complications between ABOi-KT with RIT and living ABOc-KT
(40% vs 63.3%). However, ABOi-KT patients who were treated
with RIT may have had different infection profiles. Grim
et al. retrospectively analyzed the incidence of posttransplant
infection in HLA sensitized KT or ABOi-KT treated with RIT
and compared to HLA sensitized KT without RIT. The acute
rejection rate in RIT treated KT was similar to KT without RIT
(40% vs 33%). However, posttransplant infection rate was
48.0% RIT with KT, but only 11.1% without RIT. Kamar et al.
reported that infection rate was 45.5% in KT with RIT which
was similar to KT without RIT (53.9%). Bacterial, viral and
fungal infection were observed in 36.3%, 18.2% and 16.9% in
KT with RIT, against 31.6%, 34.3% and 5.32% in KT without
RIT. Polyoma virus infection rate (64.3%) was relatively high
in RIT. Moreover, infection related-deaths were significantly
higher in RIT treated patients. This data ascertained that RIT
was associated with severe infection which causes death
rather than an increased risk of infection. Other report
confirmed earlier observation showing that the incidence of
posttransplant infection in RIT-treated recipients was similar
to RIT-untreated recipients (52.2% vs 40.2%). However, as in
earlier studies the incidences of CMV and BKV infection in
RIT-treated recipients were higher than in non RIT-treated
recipients (CMV: 16.4% vs 5.7%, BKV: 13.4% vs 8.0%).
Malignancy
It is generally accepted that immunosuppression is associated
with an increased incidence of malignancy in KT recipients
compared to the general population. However, several
studies have demonstrated that ABOi-KT did not increase
the risk of posttransplant malignancy compared with ABOc-
KT. Yamamoto et al. analyzed the risk of ABOi-KT compared
to ABOc-KT retrospectively. ABOi-KT recipients were
older than ABOc-KT recipients and all ABOi-KT recipients
received splenectomy, in this study despite increased age
and splenectomy, there was no significant difference in the
incidence of malignancy between ABOi-KT and ABOc-KT
(4.8% and 4.2%). Similarly, Hall et al. showed that 7 of 318
86  Section 1  Clinical Hemotherapy
ABOi-KT recipients experienced posttransplant cancer. The
incidence rate ratio (IRR) of cancer in ABOi-KT was identical
to that in matched control ABOc-KT (IRR: 0.99). This limited
data reassuringly indicates that ABOi-KT is not associated
with an increasing incidence of malignancy after KT. Thus, a
further analysis of long-term observations in ABOi-KT after
RIT is needed.
CONCLUSION
Since first performed over 50 years ago, ABOi-KT has become
an accepted source of KT. Reassuringly, despite lack of control
trials in ABOi-KT, more than satisfactory outcomes have been
observed in adult and pediatric recipients, in many studies
equivalent to living ABOc-KT. Preconditioning treatment of
ABOi-KT, such as antibody reduction and desensitization,
is more intensified and complicated than that of ABOc-KT.
With current protocols, the occurrence of early graft loss
and AMR are not completely abolished. Preconditioning
strategy in ABOi-KT has evolved over time. RIT has replaced
splenectomy which was once thought a crucial procedure for
ABOi-KT, although this is increasingly abandoned in favor
of IAs and IVIG. Overall, ABOi-KT is more expensive than
ABOc-KT which may restrict its adoption in resource poor
countries. We believe that a live donor ABOi-KT is a viable
alternative to waiting on deceased donor list.
Suggested reading
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Organ Transplantation. 2010;15(4):526-30.
2. Genberg H, Kumlien G, Wennberg L, Tyden G. Isoagglutinin
adsorption in ABO-incompatible transplantation. Transfusion
and Apheresis Science. 2010;43(2):231-5.
3. Haidinger M, Schmaldienst S, Körmöczi G, Regele H, Soleiman
A, Schwartz D, et al. Vienna experience of ABO-incompatible
living-donor kidney transplantation. Wiener klinische
Wochenschrift. 2009;121(7-8):247-55.
4. Jeon BJ, Kim IG, Seong YK, Han BH. Analysis of the results of
ABO-incompatible kidney transplantation: in comparison
with ABO-compatible kidney transplantation. Korean Journal
of Urology. 2010;51(12):863-9.
5. Lindberg L, Johansson SM, Liu J, Grufman P, Holgersson J. Is
there a clinical need for a diagnostic test allowing detection
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51(3):494-503.
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unanswered questions. Current Opinion in Organ Transplan­
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7. Moon HW, Yun YM, Hur M, Park JH, Lee HW, Chang SH, et al.
An experience of ABO-incompatible kidney transplantation
using plasmapheresis and anti-CD20 monoclonal antibody.
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9. Szczepiorkowski ZM, Winters JL, Bandarenko N, Kim HC,
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10. Tanabe K, Ishida H, Shimizu T, Omoto K, Shirakawa   H,
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for incompatible renal transplantation. Transfusion.
2011;51(3):454-7.
12. Tobian AA, Shirey RS, Montgomery RA, Cai W, Haas M, Ness
PM, et al. ABO antibody titer and risk of antibody-mediated
rejection in ABO-incompatible renal transplantation.
American Journal of Transplantation. 2010;10(5):1247-53.
13. Tobian AA, Shirey RS, Montgomery RA, Tisch D, Ness PM,
King KE. Therapeutic plasma exchange reduces ABO titers to
permit ABO-incompatible renal transplantation. Transfusion.
2009;49(6):1248-54.
14. Valli PV, Puga YG, Fehr T, Schulz-Huotari C, Kaup N, Güngör
T, et al. Changes of circulating antibody levels induced by
ABO antibody adsorption for ABO-incompatible kidney trans-
plantation. American Journal of Transplantation. 2009;9(5):
1072-80.
Transfusion Practice in
Heart Transplant
Hari Krishan Dhawan
Background
Heart transplant is major cardiovascular surgery performed
in heart failure patients to improve the quality of life of these
patients. As this procedure is performed in very few centers in
India, experience regarding the multidisciplinary approach
required for success of this major surgery is very limited in
our country. One of the important aspects for success of this
program is optimum transfusion support.
Pretransplant work-up
As almost always heart transplant is given ABO compatible,
ABO grouping of recipient and donor should be confirmed
from two different samples drawn separately. Red cell
antibody screen of donor and recipient should be performed
to rule out possibility of passenger lymphocyte syndrome and
to prevent transplant rejection, delayed hemolytic transfusion
reaction respectively.
Provision of leukoreduced blood
components
Preferably prestorage leukoreduced (PRBC) and apheresis
platelet concentrate and FFP should be transfused as and
when required. Leukoreduced blood components decrease
the chances of graft failure as compared to non leukoreduced
blood components by reducing the chances of HLA
alloimmunization. In our setting patient should have 8 to 10
group-matched donors available for emergency donation of
whole blood and apheresis platelets. This is more essential in
patients with Rh(D) negative blood group and patients with
multiple alloantibodies. As these surgeries happen usually
in emergency hours (because donor heart can be available
any time during day from recently deceased or brain dead
donor) blood bank should have an emergency stock of all
blood groups packed red blood cells (PRBC) on all days. At
our center we keep 6 units of PRBC, 10 FFP and 6 PC/1SDAP
ready before the surgery.
24
Transfusion requirements
during transplant
Transfusion requirements of patient during heart transplant
are usually similar to major cardiac surgery. These require­
ments changes from patient-to-patient depending on the
clinical condition of patient and drug history. According to
World literature1,2 transfusion requirement of heart trans­
plant patient is 4 PRBC (range 1-8 units), 5 FFP (1-13) and
10 random donor platelet (1-12) units or 2 apheresis platelet
concentrate. Patients undergoing transplantation after the
use of cardiac support devices require more blood. Patients
on antiplatelet drugs before surgery require more platelet
concentrates. Patients undergoing re-sternotomy or revision
surgery usually require more blood and use of aprotinin in
these settings reduces transfusion requirements as shown in
randomized studies.2 For deciding the optimum trans­fusion
support intraoperatively and postoperatively thrombo­
elastography (TEG) can be used. TEG is a convenient point
of care test for monitoring whole blood coagulation, it also
guides for the requirement of specific blood component and
optimizes the transfusion support during this major surgery.
Follow-up of transplant patients
Patients of heart transplant should be monitored at least
two weeks for development of passenger lymphocyte
syndrome (PLS) especially in cases where A, B or AB group
recipient receive heart from O group donor or cardiac
donor is having red cell alloantibodies and the recipient
red cells have corresponding antigen. PLS is an important
cause of anemia after solid organ transplant occurring 3–15
days after transplantation.3 Patients with lung and heart
transplants are at high-risk for this syndrome as volume
of lymphoid tissue transplanted is high. It is usually a self-
limiting condition, although severe cases have been treated
with immunosuppression, plasma exchange,4 and red cell
exchange. Patients should be followed up clinically and with
laboratory tests for early signs of cardiac transplant rejection.
88  Section 1  Clinical Hemotherapy
Role of photopheresis and
therapeutic plasma exchange
in cardiac transplant patients
Major advances in immunosuppression have significantly
enhanced survival and quality of life for cardiac transplant
patients, although infection, malignancies and allograft
rejection continue to threaten long-term survival. Cardiac
allograft rejection may be hyperacute (in cases of ABO or
major HLA incompatibility), acute antibody-mediated
(AMR), acute cellular (ACR), or chronic rejection (allograft
vasculopathy). ACR is the most common type of rejection and
is mediated by T cells. Extracorporeal photopheresis (ECP)
as per American Society for Apheresis (ASFA) guidelines5 is
category II therapy (accepted as second-line therapy, either
as a stand alone treatment or in conjunction with other modes
of treatment {immunosuppression}) with recommendation
1B (Strong recommendation, moderate quality evidence).
Although the mechanism of ECP is not exactly known, recent
data suggest that it decreases levels of effector T cells while
at the same time expanding regulatory T cells (Tregs). So the
ECP is used on a chronic basis as an immunomodulatory
agent. Therapeutic plasma exchange (TPE) is used to remove
donor-specific antibodies and/or inflammatory mediators
implicated in AMR. TPE’s role is in the acute setting of
rejection/desensitization.
References
1. Wegner JA, DiNardo JA, Arabia FA, et al. Blood loss and
transfusion requirements in patients implanted with a
mechanical circulatory support device undergoing cardiac
transplantation. J Heart Lung Transplant. 2000;19:504-6.
2. Prendergast TW, Furukawa S, Beyer III AJ, et al. Defining the
role of aprotinin in heart transplantation. Ann Thorac Surg.
1996;62:670-4.
3. Ramsey G. Red cell antibodies arising from solid organ
transplants. Transfusion. 1991;31:76-86.
4. Lundgren G, Asaba H, Bergstrom J, et al. Fulminating anti-A
autoimmune hemolysis with anuria in a renal transplant
recipient: A therapeutic role of plasma exchange. Clin Nephrol.
1981;16:211-4.
5. Schwartz J, Winters JL, Padmanabhan A, Balogun RA, Delaney
M, Linenberger ML, Szczepiorkowski ZM, Williams ME, Wu
Y, Shaz BH. Guidelines on the use of therapeutic apheresis in
clinical practice-evidence-based approach from the Writing
Committee of the American Society for Apheresis: the sixth
special issue. J Clin Apher. 2013;28(3):145-284.
Transfusion Practice
in Liver Transplantation
A Surekha Devi
Liver transplantation is the treatment of choice in patients
with acute or chronic end stage liver disease(ESLD),
irresectable primary liver tumors, and metabolic disorders.1
Orthotopic liver transplantation(OLT) is the replacement of
a diseased liver with a healthy liver in the normal anatomic
position. The operative procedure is extensive, complex and
technically challenging with multiple vascular transections
and anastomoses.2 In addition, OLT is associated with several
hemostatic defects that contribute to risk of massive blood
loss.2 The liver is an extremely vascular organ. The associated
coagulopathy, anemia, malnutrition and severe portal
hypertension have made this procedure more daunting and
the use of blood products almost universal.3 Hence of all solid
organ transplantations, OLT has placed the greatest demands
on clinical transfusion services.4 Although blood use steadily
declined with improved surgical and anesthesiological
techniques, better graft preservation, better intraoperative
monitoring of coagulation status and pharmacologic
treatment of fibrinolysis during the last decade, OLT still
frequently demands transfusion equal to one blood volume
(massive transfusion).5 Because the transfusion needs during
OLT are unpredictable, transfusion services must remain
prepared to effectively deliver massive transfusion support.
Liver transplant recipients present unique challenges not
only in terms of blood supply, but requirements of specialized
blood components, serologic problems, and immunologic
effects of transfusion on both the allograft and the recipient.6
Studies have observed that increased blood requirements are
associated with higher incidence of infections, drug overdose,
prolonged stay in ICU, serologic problems, and immunologic
effects of transfusion on both the allograft and the recipient,
graft rejection or graft death and patient death.1-6 However,
whether these differences in outcome are related to the
transfusion as an independent risk factor or whether the
transfusion is a marker for a technically more difficult surgery
remains unclear. Yet, bloodless liver transplantation has
been achieved in Jehovah’s witness patients.2 This subset of
patients has allowed us a distinctive opportunity to develop
strategies toward a transfusion-free environment, with
25
ultimate aim of being able to translate this practice to all
general surgical procedures.3
Contributing factors to blood loss during liver trans­
plantation can be categorized based on factors related to
preoperative and intraoperative factors.
Transfusion predictors
Preoperative Factors
Preoperative factors associated with blood loss during OLT
include liver failure, cirrhosis, cholestasis and splenomegaly.2
The liver plays a central role in hemostasis.
Two scoring systems have been used to grade the severity
of ESLD and prioritize the patients on waiting list for OLT.
Child-Turcotte-Pugh (CTP) Score
CTP score is a measure of disease severity and is classified
into CTP class A to C with a scoring system of 5–15.2
Class A: 5–6 (less severe)
Class B: 7–9
Class C: 10–15 (more severe)
Model for End-stage Liver Disease (MELD)
MELD a numerical scale, ranging from 6 (less ill) to 40 (gravely
ill), used for liver transplant patients, gives a score based on
how urgently the patient needs a liver transplant within the
next three months. Its impact on transfusion requirements
can be translated directly to the timing of the transplantation.
With longer waiting times the liver disease progresses, as
reflected in higher Child-Pugh/MELD score and thereby
increasing transfusion requirement.
Etiology of Liver Disease
Patients with chronic active hepati­tis have more advanced
disease and require more blood products than patients with
primary biliary cirrhosis.2
90  Section 1  Clinical Hemotherapy
Preoperative Hematological Parameters
A retrospective study of 300 liver transplants reported no
correlation among preoperative platelet count, aPTT, PT,
fibrinogen to that of intraoperative blood loss or transfusion
requirements.2 Blood transfusions in OLT should be based on
clinical assessment and well established transfusion protocol
rather than laboratory tests.2
Previous Abdominal Surgery
Those with previous upper abdominal surgery tend to have
vascularized adhesions which may render liver dissection
hemorrhagic thereby increasing intraoperative transfusion
requirement.2
Transjugular Intrahepatic Portosystemic
Shunt (TIPSS)
TIPSS is done in patients with liver disease for intractable
variceal bleed and refractory ascites. The benefits noted were,
it decreases the bleeding during surgery by virtue of lowered
portal pressures thereby decreasing blood requirements.2
Pre-existing Coagulopathy
It is associated with increased blood loss during surgery,
thereby increasing blood trans­fusions.2
Intraoperative Factors
Liver transplantation may be divided into three stages:
Stage I (Preanhepatic phase): Patient’s diseased liver and its
vessels are dissected free and ends with removal of diseased
organ. Blood loss occurs from transection of collaterals that
develop from portal hypertension. Pre-existing abnormalities
of clotting, platelets and fibrinolysis compound the problem.4
Stage II (anhepatic phase): Begins with implantation of the
donor liver and ends with graft reperfusion. This is dangerous
moment in the procedure when hemodynamic, metabolic
and hemostatic abnormalities can arise.4
Stage III (Postreperfusion phase): Begins with reperfusion of
grafted liver, creating hepatic arterial anastomosis, preparing
a form of biliary drainage for the new liver, obtaining good
surgical hemostasis and closing.4
During surgery, marked changes in coagulation can occur.
These may result from hemodilution, platelet consumption,
disordered thrombin regulation and fibrinolysis. Some
patients develop severe coagulopathy during the anhepatic
and early reperfusion phase of OLT.4 Transplantation of
healthy liver restores patient’s clotting function. However,
a dysfunctional graft may not immediately produce clotting
factors thereby leading to coagulopathy mandating massive/
multiple transfusions.2
Thromboelastography
Besides standard coagulation tests (i.e. PT, aPTT, fibrinogen
levels), thromboelastography (TEG) is a measurement
technique allowing rapid on-site assessment of functional
clotting status.8 TEG findings have been correlated with
intraoperative hemorrhage and coagulopathy and can
assist anesthesiologist in treating intraoperative bleeding by
identifying the cause.2 In combination with clinical bleeding
assessment, it facilitates selective use of component therapy
and specific drug treatment.8
Graft Quality
Steatosis increases cold ischemic injury and reduces the rate
of hepatic regeneration.9 The risk of primary nonfunction after
transplantation of cadaveric graft increases proportionately
with the degree of steatosis.9 Marcos et al suggested that
steatosis reduces the percentage of functioning liver.9
Increased risk of graft dysfunction is observed in fatty
infiltration of >30%, abnormal liver tests and other donor risk
factors such as high inotrope administration and donor stay
in intensive care unit (>5 days).10 Graft dysfunction further
necessitates massive/multiple transfusions.
Older donors (>60 yrs) are vulnerable to prolonged cold
ischemia and high inotrope levels, give rise to early graft
dysfunction and prolonged cholestasis.10 Such recipients are
vulnerable to receive increased transfusions.
Graft-recipient Body Weight Ratio (GRWR)
GRWR <0.8% in partial graft transplants (living donor liver
transplants and split cadaveric transplants) leads to small-
for-size syndrome (SFSS) which results in lower graft
survival.10 Such small-for size grafts depend on appropriate
blood component therapy until the graft regenerates. A
GRWR ratio of 1% is accepted to be a graft of good size.9
Graft Preservation
University of Wisconsin (UW) solution and Histidine-trypto­
phan-ketoglutarate (HTK) solution are used as primary
preserva­tion solution for liver allograft.11 Improper and
suboptimal graft preservation leads to graft dysfunction
and to severe post-transplant bleeding because of failure to
synthe­size coagulation factors which accelerates increased
blood transfusions.
Cold Ischemia Time
Length of cold ischemia time(CIT) is the length of time an
organ is preserved between procurement (organ recovery)
and transplantation. Prolonged CIT continues to have a
negative effect on liver transplant outcomes.12 CIT had a
significant effect on short-term graft failure.12 CIT should be
kept as short as possible to reduce the intraoperative RBC
transfusion requirement as prolonged CIT has negative effect
on peroperative RBC transfusion requirement.13
Surgical Technique
OLT involves explantation of native liver and replacement
with the donor liver. This is done by piggyback transplantation,
 Chapter 25  Transfusion Practice in Liver Transplantation  91
whereby the inferior vena cava is preserved and venovenous
bypass is avoided.2 Portosystemic shunting is done in
patients with liver failure in order to decrease preoperative
complications associated with portal hypertension (e.g.
bleeding varices, ascites, sepsis). This decreases blood
require­ment and operative time.2 The experience of
surgical and anesthesiologists team impacts blood loss and
transfusion requirement. Additionally, modifications in
surgical technique, including use of electrocautery, argon
beam coagulation and use of fibrin glue minimizes blood
loss thereby minimizing transfusions.2 Meticulous surgical
technique and securing hemostasis on table remains priority
for the surgeon, and indeed long operating times translate
into higher transfusion requirement.
Blood Loss
Blood loss is directly proportional to the duration of surgery.
Consumption of banked blood (no. of RBC units) reflects
the degree of blood loss. Intraoperative bleeding remains a
significant problem affecting the immediate outcome after
transplantation of liver.14 Preoperative parameters cannot
predict operative bleeding accurately and the mainstay to
prevent bleeding is a meticulous surgical technique during
the hepatectomy and correction of coagulation abnormalities
throughout the procedure.14
Hemodynamics
Central venous pressure (CVP) monitoring is an important
aspect of OLT. Patients undergo volume expansion prior
to hepatic resection to prevent bleeding complications,
but expansion increases CVP. Deliberate lowering of the
CVP during liver resection assists in bleeding control by
decreasing the blood pressure gradient over which bleeding
occurs during dissection.2 Massicotte et al showed that
maintenance of a low CVP prior to the anhepatic phase,
avoidance of plasma transfusions and restricting volume
replacement during OLT was associated with a decrease in
RBC transfusions during OLT.15
Cell Salvage
The use of cell salvage to collect and reinfuse shed,
autologous blood is common practice in OLT, when high
blood loss is anticipated.2 Autologous blood transfusion
reduces the risks associated with allogenic transfusions.
Cell salvage is not indicated in the presence of sepsis and
hepatocellular carcinoma (HCC).2 Large volume transfusion
of salvaged blood can cause postoperative hypofibrinogene­
mia, thrombocytopenia, prolonged prothrombin and partial
thromboplastin time and elevated fibrin split products.16
This cell saver induced coagulopathy can be prevented by
simultaneous platelet and cryoprecipitate transfusions along
with reinfusion of salvaged blood, as salvaged blood does not
provide platelets and fibrinogen.16,17 Saline washing of red
cells increases sodium levels and decreases potassium and
calcium levels. Hence, supplementation of potassium and
calcium is done during cell salvage.
Postoperative Factors
Primary Nonfunction (PNF) of Graft or
Delayed Graft Function
Failure of graft to function contributes to postopera­
tive bleeding, causing coagulopathy.2 Appropriate blood
component therapy is given to these patients.
Leaks at Anastomosis
Leaks at vascular suture lines or bleeding from the cut
surfaces at bowel anastomoses due to technical failures result
in postoperative bleeding which may require reintervention
and blood transfusions.2
Graft Versus Host Disease (GVHD)
GVHD can occur from transfer of donor-derived passenger
lymphocytes and mani­fests as hemolysis during 7–14 days
after transplantation.6 This is controlled by transfusion of
donor-specific RBCs.6
Sepsis
Sepsis also results in multiple blood transfusions during post-
operative period.
Thrombocytopenia
It causes bleeding which is associated with platelet consump­
tion, platelet-associated IgM and IgA antibody production,
sequestration, thrombin generation, viral infection and
ABO-incompatible GVHD.2 This further necessitates platelet
transfusions.
Drugs that minimize blood loss
Antifibrinolytics
Increased fibrinolysis is observed in some patients during
second and third phases of OLT. Various antifibrinolytic
agents like aprotinin (AP), tranexamic acid (TA) and epsilon-
amino­­caproic acid (EACA) have been used to counter
this accelerated fibrinolysis.2 Prophylactic inhibition of
hyperfibrinolysis with the biological serine protease inhibitor
AP or the synthetic lysine analog TA is common clinical
practice in OLT.18 Aprotinin has been shown to significantly
decrease blood loss, transfusion of RBC units, FFPs, platelets
and cryoprecipitate and duration of surgery.19 It also has an
effect on platelet function, anti-inflammatory properties and
the need for intraoperative ionotropes.19,20 Tranexamic acid
also decreases transfusion requirements in some but not all
studies.2
Recombinant Factor VIIa (rFVIIa)
has been shown to
improve hemostasis during liver transplantation.5 A single
92  Section 1  Clinical Hemotherapy
dose of 80 mcg/kg rFVIIa significantly reduced transfusion
requirements during OLT.5,21 A study was done where rFVIIa
was given as a bolus just before surgery to patients with
preoperative risk of high intraoperative blood loss, including
severe uncorrected coagulopathy.2 There was immediate
correction of coagulation after administration of rFVIIa and
these patients were adjusted to normal risk group without an
increased risk for thrombotic complications.2
OLT without Transfusion
Literature includes cases of orthotopic liver transplantation
(OLT) performed without transfusion of any blood products
and OLT performed safely without additional blood products
if blood loss is limited to 1600–3400 ml.2 Reports have
described OLT in Jehovah’s witness patients (for religious
reasons, Jehovah’s witnesses refuse transfusion of blood
products), who received no blood products.2 Jabbour
and colleagues continue to lead the field in performing
OLT without the use of blood products. They reported
favorable results in 27 consecutive patients who underwent
transfusion-free liver transplantation.2,3 This team used
a combination of preoperative stimulation of red cell
production using recombinant human erythropoietin and
iron and intraoperative hemodilution, cell salvage, and
tolerance to moderate anemia.3 They reported successful use
of rFVIIa in 10 patients, just prior to the incision.3 Another
team led by Olivier Detry reported successful transfusion-free
liver transplantation in 9 Jehovah’s witnesses.22
Transfusion Support
ABO Grouping
Liver transplants must be ABO compatible, because of
complement-fixing effect of ABO antibodies on endothelial
cells.6 Efforts to cross the traditional ABO barrier fall
into three categories: (i) neonatal recipients (ii) A2
organs (iii) fully ABO-incompatible organs.7 Major ABO-
incompatible liver transplants do not undergo hyperacute
rejection, but their short-term graft survival is significantly
reduced. These protocols utilize plasmapheresis, multiple
immunosuppressive agents, and sometimes splenectomy.7
An error in ABO compatibility for organs can be life-
threatening for the patient. Hence, United Network for Organ
Sharing (UNOS) instituted new requirements for additional
clerical verification of correct ABO types for transplant donors
and recipients.7 UNOS also proposed new policies to require
the performance of two separate ABO typings of each organ
donor and recipient.7
Selection of Compatible Blood Components
ABO Blood-type Selection6
Group A or B patients: Group A or B are managed with type-
specific blood and switched to group O blood, if necessary.
Group AB patients: Can receive RBCs of any group. ‘AB’
RBCs available through outdating should first be used, then
switched to group ‘A’ RBCs. After transfusing 10 units of
group ‘A’ RBCs, patient can be switched to group ‘A’ plasma.
Group O patients: Only group ‘O’ RBCs can be used in these
patients, they can receive any ABO group plasma.
Rho (D) Selection 6,7
Rh-negative patients should be provided with D- RBCs to
avoid sensitization to D. When transfusion requirements
become excessive, D-women over child-bearing age, and
adult men may be switched to D+ RBCs, provided anti-D is
not detected before transfusion. Effort should be made to
maintain D- women of child bearing age and children on
D- blood, although the risk of D alloimmunization in OLT
is low perhaps due to postoperative immunosuppression.
Such patients should be given Rh immunoglobulin (RhIg)
after administration of Rh-positive blood components. A
case of severe anti-D mediated hemolysis resulting from
liver transplantation in a O Rh+ man who received an
O Rh- liver allograft is reported.23 The female O Rh- donor had
alloantibodies against D,C, and K. This patient required two
red blood cell exchanges and intermittent red cell transfusions
negative for D,C, and K. A normalization of hemoglobin levels
and decrease in serum bilirubin occurred after a splenectomy
on postoperative Day 321.23
Selection of Red Cell Units in Patients with
Clinically Significant Alloantibodies
Clinically, significant alloantibodies are present in 6% of liver
transplant patients, due to sensitization through previous
blood transfusions.6 To minimize the risk of hemolysis,
these patients are managed by using antigen-negative units
for the first 5–10 units, switching to antigen unscreened
and/or partially matched units in the middle of the case, and
then switching back to antigen-negative units for the last
5–10 units to prevent postoperative hemolysis.6 This strategy
requires close cooperation between anesthesiologist and
transfusion services. An additional strategy would be to use
preoperative plasmapheresis to remove clinically significant
low-titer antibodies.7
Use of Autologous Blood
Preoperative autologous blood deposit, perioperative
isovolemic hemodilution, and reinfusion of salvaged blood
by cell saver during surgery minimizes allogenic transfusions.7
Specialized Blood Components
CMV-reduced Risk Components
As a result of immunosuppression, liver transplant recipients
are more susceptible to some transfusion complications such
 Chapter 25  Transfusion Practice in Liver Transplantation  93
as cytomegalovirus (CMV) infection.6 Steps should be taken
to prevent transmission of CMV through blood transfusions
to liver transplant recipients who have not been previously
infected (i.e. CMV-seronegative recipients of seronegative
donors).24 Prevention can be achieved by leukocyte reduction
of the cellular components, by selection of blood from CMV-
seronegative donors, or both.24
Leukocyte-reduced Blood Components
Transfusions before, during, and after transplant surgery
should be leukocyte reduced to reduce the risk of developing
HLA alloimmunization.24 If HLA antibodies develop in
patients waiting for an organ transplant, the risk of rejecting
the transplanted organ increases and the chance of finding a
compatible organ decreases.24
Irradiated Blood Components
Graft versus-host disease (GVHD) is a rare complication
of organ transplantation and is almost always due to donor
organ. These few cases do not support a policy of routine
irradiation of cellular blood components for solid organ
transplant recipients.6
Male only Plasma
The strategy to issue plasma from male donors for transfusion
is an effort to reduce the incidence of transfusion related
acute lung injury (TRALI), as plasma components linked to
female donors (parous women) with neutrophilic antibodies
are responsible for the majority of probable TRALI cases.25
Minor ABO-incompatible Transplants
When group O allografts are transplanted to nongroup-O
patients/nongroup-AB allografts are transplanted to group
AB patients, donor passenger lymphocytes are transferred
with the organ at the time of transplantation and produce
alloantibodies against host antigens in the recipient causing
delayed hemolysis.7 Clinically significant hemolysis caused
by anti-Jk(a), produced by passenger lymphocytes transferred
from the donor’s liver to the recipient has been reported.6
These donor derived antibodies(DDAb) develop 7–14 days
after transplantation and is heralded by the development
of positive direct antiglobulin test (DAT).6 Serum antibody
is predominantly IgG. DDAb are short lived antibodies that
persist for 2–3 weeks. Ramsey reported the incidence of
DDAb and hemolysis in OLT as 40% and 29%.6 Hemolysis
is usually mild and self-limiting. This is more likely in group
A recipients of group O livers who receive cyclosporin
or tacrolimus immunosuppression.6 In most cases the
hemolysis associated with DDAb is mild and can be treated
with transfusions.6 Several cases of hemolysis-induced acute
renal failure have been reported. In fulminant cases, red cell
exchange with donor ABO group or group O red cells, or
plasma exchange, has been performed.6
Transfusion Protocol in Minor ABO Mismatched
Liver Transplants6,7
Transfusing exclusively donor group red blood cells(RBCs)
from the beginning of surgery:
• Transfusing recipient ABO group of red cells at the
beginning of surgery, but switching to donor ABO group
of red cells after early reperfusion phase up to six weeks
in postoperative period. This replaces susceptible red cells
with cells that will not be hemolyzed. Plasma products are
transfused based on recipient ABO group to reduce the risk
of hemolysis by providing soluble ABO antigen capable
of neutralizing DDAb. After 6 weeks, if DAT and anti-A/
anti-B antibodies are negative, red cells are switched over
to recipient’s ABO group.
• Transfusing recipient group RBCs, but performing
postoperative DAT and switching to donor group RBCs if
DAT is positive.
• Transfusing recipient group RBCs until incompatibility is
noted in a crossmatch.
Transfusion Protocol During
Liver Transplantation
Criteria for replacement of blood products are as follows:
Administration of red cell units to maintain Hemo­globin
(Hb) levels at 8%; FFP (1 unit/20 kg) in case of hemorrhage
associated with International Normalized Ratio (INR) >2.0;
apheresis platelets to maintain platelet count >50 × 103/mm3
(>80 × 103/mm3 in massive transfusion) and cryoprecipitate
(1 unit/10 kg) for fibrinogen levels <100 mg/dl in case of
ongoing bleed (<120 mg/dl in massive transfusion).14,26
Hourly laboratory monitoring of hemoglobin (Hb), platelet
count, PT and fibrinogen are done throughout the surgery.
Cell saver is used to salvage blood for autologous transfusion.
In case of massive blood loss, rapid infusion pump is used
to facilitate fluid resuscitation and transfusions are given in
blood warmer. Air-warming blankets are used to maintain
body temperature.
In OLT, there is institutional variability in transfusion
practice, which mandates reassessment of the rational use of
blood products.27
When transfusions exceed more than one blood volume
within 24 hours, it is considered as massive transfusion.18
Conclusion
Multidisciplinary Approach
Proper patient selection, better preservation of graft, short
CIT, meticulous surgical technique, skilled anesthesia
practice, intraoperative autotransfusion, use of antifibrino­
lytic drugs and rFVIIa during surgery, use of TEG, and
administration of appropriate components, optimal
use of available new technologies and drugs minimizes
transfusions during OLT. Efforts to reduce intraoperative
94  Section 1  Clinical Hemotherapy
bleeding leading to limitation of the amount of blood used
are therefore desirable not only to improve results but also to
control costs, preserve the blood pool for other emergency or
routine surgeries and to decrease the program’s dependence
on blood availability. Goal should be to develop strategies
towards transfusion free environment.
References
1. Hendriks, Herman Geoge Dirk. Transfusion requirements
in orthotopic liver transplantation, Chapter 1 Introduction.
Dissertation, University of Groningen. Available from: http://
irs.ub.rug.nl/ppn/264404637 http://dissertations.ub.rug.nl/
Files/Faculties/medicine/2004/h.g.d.hendriks/C1 .pdf.
2. Richard K Spence, Julie Maurer. Transfusion requirements in
liver transplantation – e medicine updated: May 25, 2006.
3. Nicolas Jabbour, Singh Gagandeep, Rodrigo Mateo, Linda
Sher, Yuri Genyk, Rick Selby. Transfusion free surgery: Single
Institution experience of 27 consecutive liver transplants in
Jehovah’s witnesses. Journal of American College of Surgeons.
2005;3:412-7.
4. Walter H Dzik. Solid organ transplantation. In: Lawrence D
Petz, Scott N Swisher, Steven Kleinman, Richard K Spence,
Ronald G Strauss (Eds): Clinical Practice of Transfusion
Medicine, 3rd edn. New York: Churchill Livingstone; 1996. pp.
792-802.
5. Herman GD Hendriks, Karina Meijer, Joost Th MDE Wolf, Ids J
Klompmaker, Robert J Porte, Peter Jan De Kam, et al. Reduced
transfusion requirements by recombinant factor VIIa in
orthotopic liver transplantation: A pilot study. Transplantation.
2001;71:402-5.
6. Darrel J Triulzi. Transfusion support in liver transplantation.
Current Hematology Reports. 2004;3:444-9.
7. Glenn Ramsey, Paul D. Mintz. Transfusion practice in solid
organ transplantation. In: Mintz PD (Ed). Transfusion Therapy:
Clinical Principles and Practice, 2nd edn. Bethesda, MD:
American Association of Blood Banks press; 2005. pp. 265-74.
8. McNicol PL, Liu G, Harley ID, McCall PR, Przybylowski
GM, Bowkett J, et al. Patterns of coagulopathy during liver
transplantation: experience with the first 75 cases using
thromboelastography. Anaesth Intensive Care. 1994;22:659-65.
9. Broering DC, Sterneck M, Rogiers X. Living donor liver
transplantation. J Hepatol. 2003;38:S119-35.
10. Heaton N. Small-for-size liver syndrome after auxillary and
split.liver transplantation: donor selection. Liver Transpl.
2003;9: S26-8.
11. Richard S Mangus, A Joseph Tector, Avinash Agarwal, Rodrigo
Vianna, Philip Murdock, Jonathan A Fridell. Comparison
of histidine-tryptophan-ketoglutarate solution (HTK)
and University of Wisconsin solution (UW) in adult liver
transplantation. Liver Transpl. 2006;12:226-30.
12. Erick B Edwards, Richard B Freeman. Effect of cold ischemia
time in liver transplantation assessed. UNOS News Bureau(804)
782-4730, May 18, 2004. Available from: newsroom@unos.org .
13. HGD Hendriks. Transfusion requirements in liver
transplantation, Chapter8–Conclusions. Available from:
http://www.ub.rug.nl/eldoc/dis/medicine/h.g.d.hendriks/
conclusions_and_recommendations.pdf.
14. Mor E, Jennings L, Gonwa TA, Holman MJ, Gibbs J, Solomon
H, et al. The impact of operative bleeding on outcome in
transplantation of the liver. Surg Gynecol Obstet. 1993;176:219-
27.
15. Luc Massicotte, Serge Lenis, Lynda Thibeault, Marie-Pascale
Sassine, Robert F Seal, Andre Roy. Effect of low central venous
pressure and phlebotomy on blood product transfusion
requirements during liver transplantations. Liver Transpl.
2005;12:117-23.
16. Laurence A. Sherman, Glenn Ramsey. Solid-organ transplan­
tation. In: Ennio C Rossi, Toby L Simon, Gerald S Moss, Steven
A Gould (Eds): Principles of Transfusion Medicine, 2nd edn.
Baltimore: Williams and Wilkins; 1996. pp. 635-7.
17. Constance F. Danielson. Transfusion support and complicating
coagulopathies in solid organ transplantation. In: Hackel E,
Aubuchon JP (Eds): Advances in transplantation, 1st edn.
Bethesda, MD: American Association of Blood Banks; 1993. p.
43-64.
18. Brigitte E.Ickx, Philippe J.van der Linden, Christian Melot,
Walter Wijns, Luc de Pauw, Jean Vandestadt et al. Comparison
of the effects of aprotinin and tranexamic acid on blood loss
and red blood cell transfusion requirement during the late
stages of liver transplantation. Transfusion. 2006; 46: 595-605.
19. Victor W Xia, Radolph H. Steadman. Antifibrinolytics
in orthotopic liver transplantation: Current status and
controversies. Liver Transpl. 2005;11:10-8.
20. Findlay JY, Kufner RP. Aprotinin reduces vasoactive medication
use during adult liver transplantation. J Clin Anaesth. 2003;15:
19-23.
21. Lodge JP, Jonas S, Jones RM, Olausson M, Mir-Pallardo J, Soefelt
S, et al. Efficacy and safety of repeated perioperative doses of
recombinant factor VIIa in liver transplantation. Liver Transpl.
2005;11:973-9.
22. Oliver Detry, Arnaud De Roover, Jean Delwaide, Abdour
Kaba, Jean Joris, Pierre Damas, et al. Liver transplantation in
Jehovah’s witnesses. Transplant International. 2005;18:929-36.
23. Fung MK, Sheikh H, Eghtesad B, Lopez-Plaza I. Severe
hemolysis resulting from D incompatibility in a case of ABO-
identical liver transplant Transfusion. 2004;44:1635-9.
24. Ted Eastlund. Tissue and Organ Transplantation and the
Hospital Tissue Transplantation Service. In: John D Roback,
Martha Rae Combs, Brenda J Grossman, Christopher D Hillyer
(Eds). Technical Manual, 16th edn. Bethesda, MD: American
Association of Blood Banks; 2008. pp. 850-2.
25. Anne F Eder, Ross Herron, Annie Strupp, Beth Dy, Edward pp.
Notari, Linda A Chambers, et al. Transfusion-related acute lung
injury surveillance (2003-2005) and the potential impact of the
selective use of plasma from male donors in the American Red
Cross. Transfusion. 2007;47:599-607.
26. Emilio Ramos, Antonia Dalmau, Antonio Sabate, Carmen
Larma, Laura Llado, Juan Figueras, et al. Intraoperative red
blood cell transfusion in liver transplantation: Influence on
patient outcome, prediction requirements, and measures to
reduce them. Liver Transpl. 2003;9:1320-7.
27. Yves Ozier, Fabienne Pessione, Emmanuel Samain, and
Francoise Courtois. Institutional variability in transfusion
practice for liver transplantation. Anaesth Analg. 2003;97:
671-9.
 Massive Transfusion
Massive Transfusion Protocol
Kanchan Bhardwaj, Rajni Bassi
Mortality from hemorrhagic shock is number one cause
of civilian and military trauma victims.1 Most recently,
there has been significant interest in protocolization of this
transfusion process. Studies demonstrate improved patient
outcome with implementation of a massive transfusion
protocol (MTP) when compared to physician/lab driven
resuscitation.2-5 Multiple military and civilian trauma studies
of massive transfusion protocols suggest that a 1:1:1 ratio of
packed red blood cells (PRBC) to fresh frozen plasma (FFP)
and platelets (PLT) is associated with the best outcomes.2-4,6
This improved mortality has been attributed to reduced
time to first transfusion of products, thus addressing the
fundamental problem of coagulopathy. MTP is activated by a
clinician in response to a patient that is experiencing massive
bleeding. The responsible clinician activates the MTP either
by phone or any other mechanism. Once a patient is in the
protocol, the blood bank is able to insure rapid and timely
availability of blood components to facilitate resuscitation.
The need for MTP may be encountered in either surgical or
medical emergencies where life-threatening hemorrhage is
confronted. Surgical control of hemorrhage (damage con­
trol surgery) and timely application of volume resuscitation
with fluids and blood components remains the cornerstone
of treatment.7 Specific injury patterns that should prompt
consideration for implementation of a MTP include liver
laceration with hemorrhage, emergent abdominal aortic
aneurysm, pelvic fracture with overwhelming blood loss,
massive gastrointestinal hemorrhage, and coronary artery
bypass grafting. Once definitive control of bleeding has been
achieved, a restrictive approach to blood product transfusion
is preferred because of the well-known risks and negative
outcomes of transfusion such as multiple organ failure,
systemic inflammatory response syndrome, TRALI, increased
infection, and increased mortality.8,9
RATIONALE
Transfusion of RBCs, plasma, and platelets in a similar
proportion as whole blood may minimize the effects of
26
dilutional coagulopathy and hypovolemia.10 In a survey
of members of EAST (Eastern Association for the Surgery
of Trauma), 85% of US trauma centers have a MTP.11 The
present data do show that having a MTP has better outcomes
than not having one.11 
DEFINITION OF MASSIVE TRANSFUSION8
The traditional definition of massive transfusion is
• 20 units RBCs in 24 hours, corresponding to approximately
1 blood volume in a 70 kg patient
• ≥ 10 units RBCs in 24 hours12 
• Loss of 0.5 blood volume within 3 hours
• Use of 50 units of blood components in 24 hours
• Use of 6 units RBCs in 12 hours
• Practicaly requirement for > 4 RBC units in 1 hour with
ongoing need for transfusion,
• or blood loss > 150 ml/min with hemodynamic instability
and need for transfusion are reasonable definitions in the
setting of a MTP situation.
The variability in defining massive bleeding may result in
variability initiating a MTP and another reason why having
a MTP vs. not may result in early resuscitation and better
outcome.
PHYSIOLOGY OF HEMORRHAGIC SHOCK
Major trauma causes tissue injury and hemorrhage. Shock
occurs due to decreased circulating volume, cardiac output
and oxygen delivery which is compounded by the associated
inflammation, acidosis, and tissue hypoperfusion. Environ­
mental exposure, initial resuscitation with unwarmed fluid
and deranged thermoregulation result in hypothermia.
Aggressive fluid resuscitation can cause hemodilution with
decreased oxygen carrying capacity, decreased oxygen deli­
very, and dilution of substrate and enzymatic coagulation
factors. Progressive coagulopathy leads to further hemorrhage
and shock. Refractory coagulopathy, progressive hypothermia
and persistent metabolic acidosis lead to end stage shock and
96  Section 1  Clinical Hemotherapy
death. Prevention and control of coagulopathy is the goal
of MTP in the hope of preventing further hemorrhage after
trauma.1
COAGULOPATHY OF HEMORRHAGE AND
MASSIVE TRANSFUSION
Patients may have a complex coagulation disorder involving
dilution of pro-coagulant, anti-coagulant, pro-fibrinolytic
and anti-fibrinolytic factors.13-15  Acute coagulopathy of
trauma appears to be due to activation of anticoagulant and
fibrinolytic pathways. Shock and hypoperfusion are the key
drivers of acute coagulopathy of trauma. Hypoperfusion
increases the expression of thrombomodulin on endothelium
which then complexes with thrombin.16 This reduces the
amount of thrombin available to produce fibrin and increases
activated protein C. In the obstetric patient, massive bleeding
may routinely consume fibrinogen despite available thrombin,
hence differences might exist in mechanism of coagulopathy
in different patient populations. In trauma, hypothermia and
acidosis play a key role by reducing thrombin generation
due to altered enzyme kinetics. Both coagulation factors
and platelets are reduced by hemodilution. Platelet count is
higher than predicted due to release of platelets sequestered
in the spleen and lungs. Antithrombin activity decreases to
less than 30% of normal with extensive hemodilution.17 A
threshold level of fibrinogen 1g/L (100 mg/dL) is reached
after losing 150% of circulating blood volume as opposed to
200% for clotting enzymes.18 Systemic hyperfibrinolytic states
may be seen in up to 20% of trauma patients.19
PREDICTING NEED FOR MTP
Approximately 3-5% of civilian adult trauma patients receive
massive transfusion.20 Early identification of patients requir­
ing MTP has been evaluated by assigning a value of 0 or 1
to the following four parameters: penetrating mechanism,
positive FAST for fluid (focused assessment sonography in
trauma), arrival blood pressure <90 mm Hg, and arrival pulse
>120 bpm. A score of 2 or more is considered positive.21 The
score is 75% sensitive and 85% specific. FAST examination
identifies whether there is free fluid within the peritoneum,
which could indicate organ rupture and internal bleeding
(Table 11 and Table 222).
ADJUNCTS TO MTP
Hypothermia, acidosis, shock, coagulopathy, and failure to
control surgical bleeding contribute to mortality after trauma.
In addition to a MTP, all efforts must be directed towards
“damage control resuscitation” including control of surgical
bleeding and correction of hypothermia, acidosis, shock, and
coagulopathy. Implementation of clinical practice guidelines
have led to improved survival in trauma.23
HYPOTHERMIA
Hypothermia is a frequent pathophysiologic consequence
of severe injury and subsequent resuscitation.24 Gentilello
classified the severity of hypothermia in the trauma patient
as mild (36–34°C), moderate (33.9–32°C), and severe
(below 28°C).24 Body temperatures less than 33°C produce
a coagulopathy that is functionally equivalent to factor
deficiency states seen when coagulation factor concentrations
are less than 50%.24 Thrombin generation on platelets is
reduced by 25% at 33°C. The average size of aggregates formed
by thrombin-activated platelets was decreased by 40% at
33°C and platelet adhesion was reduced by 33%.25 Adverse
clinical effects such as cardiac dysrhythmias, reduction in
cardiac output, increase in systemic vascular resistance, and
a left shift in the oxygen-hemoglobin saturation curve have
been described. Mortality rates as high as 100% are seen in
patients with severe hypothermia and severe injury. The most
significant effect of hypothermia in trauma is coagulopathic
bleeding due to prolonged clotting cascade enzyme reactions,
dysfunctional platelets, and fibrinolysis.26,27
REWARMING STRATEGIES
Rewarming strategies initiated in the emergency department
and operating room are aggressively continued in the
intensive care unit. Strategies include passive and active
external rewarming and active core rewarming.
Passive External Rewarming
Passive external rewarming involves removing blood or
saline-soaked dressings or blankets, increasing ambient
room temperature, and decreasing air flow over the patient
by keeping the room doors shut.
Active External Rewarming
Active external rewarming devices include fluid/air circul­
ating blankets, aluminum space blankets and overhead
radiant warmers. Convective-air and aluminum space blan­
kets placed over the patient provide greater heat exchange by
creating a 43°C microenvironment around the patient, which
effectively stops heat loss. Superior warming is achieved
when standard cotton blankets are placed over these blankets
and the edges secured, although this limits patient access.
Head covering is of prime importance; because significant
vasoconstriction does not occur in scalp vessels, and as
much as 50% of radiant heat loss occurs from the neck up.6
Aluminized caps are effective warmers, but their use is
limited in head injured patients with intracranial pressure
(ICP) monitors. The overhead radiant warmers when aimed
directly onto vasoconstricted skin may cause inadvertent
burns; yet when directed over a blanket, they provide no
direct heat exchange to the patient. During laparotomy, it

Table 1: Massive transfusion protocol adult1
is recommended that covering exposed bowel with moist
towels be avoided because it can increase evaporative heat
loss by nearly 250%.24 Dry towels or plastic bags are superior.
Active Core Rewarming
The hypothermic trauma patient requires active core
rewarming which may include airway rewarming, heated
body cavity lavage, heated intravenous fluids, continuous
arteriovenous rewarming (CAVR), and extracorporeal
circulatory rewarming. Humidified ventilator circuits can be
warmed to 41°C.
Chapter 26  Massive Transfusion Protocol  97
Use of warmed intravenous fluids is one of the simplest
and most effective means of providing heat to the core
in patients requiring massive fluid resuscitation. Current
fluid warmer technology allows large volumes of warmed
fluids to be infused quickly at 42°C, the current standard
recommended by the American Association of Blood Bank.28
Blood-warming methods include surface-contact warmers,
counter-current warmers, and heated-saline admixture.29 In-
line microwave blood-warming technology (in development)
has been shown to heat blood safely to 49°C and shows great
promise for the future.30
98  Section 1  Clinical Hemotherapy

Table 2: Recommendation of massive transfusion protocol22

RECOMMENDATIONS
•  Level 1: None
•  Level 2:
   –  Administer blood products in a ration of 1:1:1 (PRBC: FFP: PLT).
   –  In patients requiring massive transfusion of blood products, minimize crystalloid resuscitation to prevent dilutional coagulopathy.
   –  Platelet transfusions are indicated in the following situations:
     -  Neurosurgical procedures or traumatic brain injury (TBI) with Plt count<100 × 109/L.
     -  Surgical/obstetric patients with microvascular bleeding and Plt count<50 × 109/L.
     -  Any surgical patient with Plt count< 20 × 109/L.
   –  FFP (10–15 mL/kg) is indicated in the following situations:
     -  Hemorrhage with elevated PT or PTT (> 1.5 times normal).
     -  Urgent reversal of warfarin therapy (see “Warfarin Reversal Guideline”)
   –  Cryoprecipitate should be administered in the following situations:
     -  Hemorrhage with fibrinogen concentrations <100 mg/dL
     -  Bleeding patients with von Willebrands disease.
   –  Tranexamic acid should be considered in patients with significant hemorrhage presenting within 3 hours of injury.
•  Level 3:
   –  Consider the massive transfusion protocol (MTP) in the presence of:
    - Systolic blood pressure ≤ 90 mm Hg
    - Heart rate ≥ 120 beats per minute (bpm)
     -  Positive focused sonography for trauma (FAST) exam
    - pH ≤ 7.24
   – Consider MTP implementation if transfusing ≥ 4 units of PRBCs over 1 hour or expected ≥ 10 units over 24 hours (more than one total
blood volume).
   –  Maintain platelet counts above 100 × 109/L during times of active hemorrhage.
   –  Correct moderate and severe hypothermia (<34oC)
     -  Place convective-air or aluminum space blankets over the patient.
     -  Use humidified mechanical ventilator circuits warmed to 41oC.
     -  Use fluid warmers for the infusion of fluids at 42oC.
     -  For refractory hypothermia, consider pleural/peritoneal lavage, or arteriovenous rewarming.
   –  Consider bicarbonate administration when pH < 7.2

EVIDENCE DEFINITIONS
• Class I: Prospective randomized controlled trial.
• Class II: Prospective clinical study or retrospective analysis of reliable data. Includes observational, cohort, prevalence, or case control studies
• Class III: Retrospective study includes database or registry reviews, large series of case reports, expert opinion.
• Technology assessment: A technology study which does not lend itself to classification in the above-mentioned format. Devices are evaluated in terms
of their accuracy, reliability, therapeutic potential, or cost effectiveness.
LEVEL OF RECOMMENDATION DEFINITIONS
• Level 1: Convincingly justifiable based on available scientific information alone. Usually based on Class I data or strong Class II, evidence if randomized
testing is inappropriate. Conversely, low quality or contradictory Class I data may be insufficient to support a Level I recommendation.
• Leve 2: Reasonably justifiable based on available scientific evidence and strongly supported by expert opinion. Usually supported by Class II data or a
preponderance of Class III evidence.
• Level 3: Supported by available data, but scientific evidence is lacking. Generally supported by Class III data. Useful for educational purposes and in
guiding future clinical research.

In continuous arteriovenous rewarming (CAVR),31 8.5
French femoral arterial and venous catheters are placed
percutaneously, an extracorporeal arteriovenous circuit
that uses the heating mechanism of a counter-current fluid
warmer are created. This is more effective in comparison with
traditional warming techniques.29
ACIDOSIS
The association between high lactate levels and increasing
risk of death was with metabolic acidosis as demonstrated
by arterial pH, lactate, and base deficit clearance.32 The
deleterious effects of acidosis on the cardiovascular system
include decreased cardiac contractility and cardiac output,
vasodilation and hypotension, decreased hepatic and renal
blood flow, bradycardia, and increased susceptibility to
ventricular dysrhythmias.33 Acidosis directly reduces the
activity of the extrinsic and intrinsic coagulation pathways as
measured by PT and PTT and also diminishes platelet function
as measured by platelet aggregation and platelet factor III
release.24 These adverse effects are generally not seen until pH
decreases below 7.2.33 Therapy for metabolic acidosis remains
directed toward correcting the underlying hypoperfusion.
Resuscitation endpoints include normalization of arterial pH,

base deficit, and lactate. Bicarbonate administration should
be deferred until the pH persists below 7.2, despite optimal
fluid loading and inotropic support.34
TRANEXAMIC ACID
Tranexamic acid is an antifibrinolytic agent that has
historically been shown to decrease the need for blood
transfusions in patients undergoing elective surgery. In
2010, a multinational randomized, double-blind placebo-
controlled trial (CRASH-2) showed that the tranexamic acid
group had a significantly lower all-cause mortality at 28
days.35
RESUSCITATION IN PATIENTS REQUIRING
MASSIVE TRANSFUSION
Early control of hemorrhage is essential to limit consumptive
coagulopathy and thrombocytopenia, and decrease usage
of blood. Limiting the use of isotonic crystalloid will help
prevent dilutional coagulopathy. Limiting the use of
0.9% saline may prevent further acidosis. Hypotensive
resuscitation (systolic blood pressure 80–100 mm Hg) until
hemorrhage is controlled is generally recommended, unless
there is concern for traumatic brain injury. Early use of
plasma, platelets as well as RBCs is necessary in an attempt to
improve survival with massive transfusion. Fibrinogen levels
above 1.5 g/L are targeted.36 These levels can be achieved with
cryoprecipitate 50 mg/kg or fibrinogen concentrate doses of
3–4 g.36 Above base deficit and lactate levels are monitored to
assess adequacy of resuscitation in restoring oxygen delivery
and tissue perfusion. Electrolyte abnormalities caused by
dilution and transfusion should be corrected: hyperkalemia
from large volume of banked RBCs, hypocalcemia from
citrated anticoagulants, and Na and Cl abnormalities from
crystalloid resuscitation. Evidence is growing for use of point
of care coagulation testing to guide hemostatic therapy
(thromboelastography, thromboelastometry, Sonoclot)6,37
Standard coagulation tests such as PT, PTT, INR, platelet
count, and fibrinogen usually require 30–60 minutes for
results to be available. For massive transfusion patients
requiring acute interventions, results of standard coagulation
tests may not be an accurate reflection of coagulation
function. Measurement of platelet count and fibrinogen only
provides absolute amounts, not functional activity and may
over estimate the levels.
THROMBOELASTOGRAPhY (TEG) AND
ROTATIONAL THROMBOELASTOMETRY
(ROTEM)
Two available viscoelastic whole-blood assays measured at
37°C give an in vitro measure of clotting. These hemostatic
assays provide a graph of the displacement caused by
Chapter 26  Massive Transfusion Protocol  99
viscoelastic changes in whole blood as clotting progresses and
fibrin strands form between the cup and pin. Clot strength is
decreased by hypofibrinogenemia, thrombocytopenia, low
F XIII level, or reduced thrombin generation. Rapid TEG
(coagulation is initiated by the addition of tissue factor) gives
faster results compared to kaolin TEG. ROTEM, EXTEM
(uses recombinant tissue factor to activate coagulation)
and FIBTEM (measures contribution of fibrinogen to clot
strength) allow differential diagnosis of thrombocytopenia
and hypofibrinogenemia. Higher hemoglobin levels reduce
TEG/ROTEM signals. 
Application
Hyperfibrinolysis was identified in the most severely injured
patients, and correlated with mortality.38,39 Kashuk et al.
demonstrated primary fibrinolysis in 34% of patients requiring
massive transfusion using TEG.40 Early administration of
an anti-fibrinolytic agent (tranexamic acid) to adult trauma
patients resulted in reduced all-cause mortality in the
treatment group, reduced mortality from bleeding and a
safe side effect profile in bleeding trauma patients.41 It is
recognized, however, that severely injured patients who pre­
sent fibrinolysis have increased mortality14,42 so the efficacy
of tranexamic acid in such patients is questionable. TEG/
ROTEM assays may signal the selection of patients who would
most benefit from this intervention.
FACTOR rVIIa AND MTP
Evidence shows benefit for rFVIIa, especially in coagulopathic
trauma patients, however this remains an off-label use
and controversial. rFVIIa is not a first-line therapy for
hemorrhage. Adequate fibrinogen levels (e.g. > 1.5–2 g/L) and
platelet count (e.g. > 50–100 × 109/L) are necessary for drug
efficacy. Control of surgical bleeding and normalizing acid-
base status and core temperature are of obvious importance.
In a meta-analysis of patients with body trauma, use of rFVIIa
reduced ARDS and transfusion requirements, but had no
effect on mortality or thromboembolism.43 Because of the
risk of serious adverse effects, treatment with rFVIIa must be
individualized based on a risk-benefit analysis.
COMPLICATIONS OF MTP
Blood transfusion is an independent predictor of multi-organ
failure in a retrospective and prospective study of trauma
patients with injury severity score > 15 and survival greater than
24 hours.9 There is a 2–6 fold increase in SIRS, 4 fold increase
in ICU admission and mortality in transfused patients. One
study showed that transfusion was an independent predictor
of death in non-operative blunt trauma and the risk of death
increased with each additional RBC unit.44 Multiple studies
have linked transfusion to increased development of and
mortality from ARDS. TRALI occurs with a 1:5000 incidence in
100  Section 1  Clinical Hemotherapy
the general transfusion population and is likely increased in
massive transfusion, although the extent is unknown. TRALI
is likely under-diagnosed after severe injury. It is difficult to
differentiate TRALI from transfusion associated circulatory
overload or other causes of lung injury in trauma. For non-
massively transfused trauma patients, plasma administration
is associated with increased complications (ARDS, multiple
organ dysfunction, pneumonia, and sepsis) with no improve­
ment in survival.45 Despite the claim that high ratio MTP
reduces dilutional coagulation, some clotting factors do get
diluted by the shear volume of components and the limited
availability of factors in plasma may not be sufficient to
overcome this defect. As such, the early and sometime the
sole use of coagulation factors and fibrinogen concentrates is
being adopted in European centers. The use of these agents
in the US is off label and many are not FDA approved. Firm
data on efficacy and safety of this practice is needed and is
slowly accruing.
MTP AFTER CONTROL OF HEMORRHAGE
A restrictive approach to blood product utilization is advo­
cated once bleeding is stopped and the patient is stable.9
RBC transfusion is generally indicated for hemoglobin
< 7 g/dL. For hemoglobin above 7 g/dL, hypovolemia is
treated with IV fluids. Signs and symptoms of impaired
oxygen delivery and decreased tissue perfusion are closely
monitored. RBC transfusion is generally required for
persistent base deficit, lactic acidosis, signs of end organ
ischemia, and low mixed venous oxygenation.
MTP IN PEDIATRICS
Massive transfusion for pediatric trauma patients generally
follow similar principles as in adults.46 An age and weight-
based approach is required. Paterson used a MTP protocol for
a 5-year-old child with massive bleeding during neurosurgery
(estimated loss of between five and six blood volumes over
1.5 hours.47 The first blood volume was replaced with
crystalloid (40 ml/kg), RBCs (30 ml/kg) and plasma (20 mL/
kg FFP). RBCs, plasma, platelets, cryoprecipitate and rFVIIA
were used subsequently. The child survived. Further studies
are necessary to validate use of MTP in children.
SUMMARY
Massive hemorrhage and resuscitation can result in refractory
coagulopathy if not aggressively treated. The use of MTPs
facilitate rapid availability of components. Increased ratios
of plasma and platelets to RBCs and their timely adminis­
tration improve outcome in trauma, decrease coagulopathy
and transfusion requirements. Large volumes of plasma are
required to correct coagulopathy, so early administration is
presumably more efficacious. The approach would be diffe­
rent when specific factor concentrates are used.48 Viscoelastic
assays may allow for goal directed therapy in coagulopathy
of trauma and massive transfusion including the use of
antifibrinolytics when appropriate. Single agent therapy
such as rFVIIa may have a role in coagulopathic trauma
patients but safety is still a concern. A restrictive transfusion
strategy should be adopted once hemorrhage is controlled to
minimize unnecessary exposure to blood.
The MTP practice is still fraught with many unresolved
issues such as use of fibrinogen and/or prothrombin complex
concentrate and blunt vs penetrating trauma. Understanding
the mechanism of hemorrhage is not universal and is
different in the obstetrical population as it is in pediatric
or cardiovascular patients. This may add to the limitation
of universal adoption of a single ratio driven MTP. Well
designed, prospective randomized trials are required to
determine optimal transfusion ratios and timing of blood
component administration.
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12. Repine TB, et al. The use of fresh whole blood in massive human packed red blood cells. Crit Care Med. 1995;23:1243-
transfusion. J Trauma. 2006;60(6 Suppl):S59-69.  50.
13. Bolliger D, Görlinger K, Tanaka KA. Pathophysiology and 31. Gentilello LM, Cortes V, Moujaes S, et al. Continuous
treatment of coagulopathy in massive hemorrhage and arteriovenous rewarming: Experimental results and thermo­
hemodilution. Anesthesiology. 2010;113(5):1205-19.  dynamic model simulation of treatment for hypothermia. J
14. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie Trauma. 1990;30:1436-49.
RC, Pittet JF. Acute coagulopathy of trauma: hypoperfusion 32. Abramson D, Scalea T, Hitchcock R, et al. Lactate clearance and
induces systemic anticoagulation and hyperfibrinolysis. J survival following injury. J Trauma 1993;35:584-9.
Trauma. 2008;64(5):1211-7.  33. Wildenthal K, Mierzwaiak DS, Myers RW, et al. Effects of acute
15. Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie lactic acidosis on left ventricular performance. Am J Physiol.
RC, Pittet JF. Acute traumatic coagulopathy: initiated by 1968;214:1352-9.
hypoperfusion: modulated through the protein C pathway? 34. Wilson RF Shock. In: Critical Care Manual: Applied Physiology
Ann Surg. 2007;245(5):812-8.  and Principles of Therapy. Philadelphia: FA Davis; 1992:267.
16. Frith D, Brohi K. The acute coagulopathy of trauma shock: 35. CRASH-2 trial collaborators. Effect of tranexamic acid on
Clinical relevance. Surgeon 2010;8:159-63.  death, vascular occlusive events, and blood transfusion in
17. Bolliger D, Szlam F, Molinaro RJ, Rahe-Meyer N, Levy JH, trauma patients with significant haemorrhage: a randomized,
Tanaka K. Finding the optimal concentration range for placebo-controlled trial. Lancet, 2010;376:23-32.
fibrinogen replacement after severe haemodilution: an in vitro 36. Rossaint R, et al. Management of bleeding following major
model. Br J Anaesth. 2009;102:793-9.  trauma: an updated European guideline. Crit Care 2010;14:R52.
18. Hiipala, et al. Hemostatic factors and replacement of major 37. Johansson PI, et al. Thromboelastography and thromboelasto­
blood loss with plasma-poor red cell concentrates. Anesth metry in assessing coagulopathy in trauma. Scand J Trauma
Analg. 1995;81:360-5. Resusc Emerg Med. 2009;17:45.
19. Kashuk JL, et al. Primary fibrinolysis is integral to the 38. Levrat, et al. Evaluation of rotation thromboelastography
pathogenesis of the acute coagulopathy of trauma. Ann Surg. for the diagnosis of hyperfibrinolysis in trauma patients. Br J
2010;252(3):434-42. Anaesth. 2008;100:792-7.
20. Nunez TC, Young PP, Holcomb JB, Cotton BA. Creation, 39. Carroll RC, et. Al. Early evaluation of acute traumatic
implementation, and maturation of a massive transfusion coagulopathy by thromboelastography. Trans Res. 2009;154:34-
protocol for the exsanguinating trauma patient. J Trauma. 9.
2010;68:1498-505. 40. Kashuk JL, et al. Postinjury coagulopathy management: Goal
21. Cotton BA, Dossett LA, Haut ER, et al. Multicenter validation directed resuscitation via POC thromboelastography. Ann
of a simplified score to predict massive transfusion in trauma. J Surg. 2010;251(4):604-14.
Trauma. 2010;69 Suppl 1:S33-9. 41. Effects of tranexamic acid on death, vascular occlusive events,
22. Massive Transfusion for Coagulopathy and Hemorrhagic Shock, and blood transfusion in trauma patients with significant
http://www.surgicalcriticalcare.net/Guidelines/massive%20 haemorrhage (CRASH-2): a randomized, placebo-controlled
transfusion%20protocol%202012. trial. Lancet, 2010.
23. Simmons JW, White CE, Eastridge BJ, Mace JE, Wade CE, 42. Hess JR, Brohi K, Dutton RP, et al. The coagulopathy of trauma:
Blackbourne LH. Impact of policy change on US Army combat a review of mechanisms. J Trauma. 2008;65:748-54.
transfusion practices. J Trauma. 2010;69 Suppl 1:S75-80. 43. Yank V, et al. Systematic review: benefits and harms of in-
24. Gentilello LM, Jurkovich GJ. Hypothermia. In: Ivatury RR, hospital use of recombinant factor VIIa for off-label indications.
Cayten CG, eds. The Textbook of Penetrating Trauma. Ann Intern Med. 2011;154:529-40. 
Baltimore: Williams & Wilkens; 1996;995-1005. 44. Robinson WP, et al. Blood transfusion is an independent
25. Luna GK, Maier RV, Pavlin EG, et al. Incidence and effect predictor of increased mortality in non-operatively managed
of hypothermia in seriously injured patients. J Trauma. blunt hepatic and splenic injuries. J Trauma. 2005;58(3). 
1987;27:1014-8. 45. Inaba K, et al. Impact of plasma transfusion in trauma patients
26. Frank S, Beattie C, Christopherson R, et al. Unintentional who do not require massive transfusion. J Am Coll Surg.
hypothermia is associated with post operative myocardial 2010;210:957-65. 
ischemia. Anesthesiology 1993;78:468-72. 46. Dehmer JJ, Adamson WT. Massive transfusion and blood
27. Britt L, Dascombe W, Rodriguez A. New horizons in product use in the pediatric trauma patient. Seminars in
management of hypothermia and frostbite injury. Surg Clin Pediatric Surgery 2010;19:286-91. 
North Am. 1991;71:345-70. 47. Paterson NA. Validation of a theoretically derived model for
28. American Association of Blood Banks. Technical Manual. the management of massive blood loss in pediatric patients—a
Standards for Blood Banks and Transfusion Services. 18th edn. case report. Paediatr Anaesth. 2009;19:535-40.
Bethesda, MD: American Association of Blood Banks; 1998. 48. Schochl H, et al. Goal-directed coagulation management of
29. Iserson K, Huestis D. Blood warming: Current applications and major trauma patients using thromboelastometry (ROTEM®)-
techniques. Transfusion 1991;31:558-71. guided administration of fibrinogen concentrate and
30. Pappas C, Paddock H, Goyette P, Grabowy R, Connolly R, prothrombin complex concentrate. Critical Care 2010;14:R55.
Schwaitzberg S. In-line microwave blood warming of indate
Massive Transfusion in
Obstetrical Hemorrhage
Sangeeta Pathak
PRINCIPLE
The massive transfusion protocol (MTP) for obstetrical
Hemorrhage is intended for antepartum, intrapartum
or postpartum patients deemed candidates based on
requirement for massive blood volume replacement. This
protocol has been prepared to meet the special needs of the
obstetrical hemorrhage patient.
DEFINITION
Any blood loss occurring in the peripartum period,
revealed or concealed, that is likely to endanger life.
Physiological and hematological changes induced by
pregnancy can hide signs of hypovolemic shock and patient
can collapse suddenly.
Obstetric hemorrhage remains a major cause of maternal
mortality in the UK, with substandard management identified
as a contributor in 80% of the cases.1 It is estimated that
there are more than 4000 cases of severe hemorrhage each
year in the UK. The majority of these cases will need blood
transfusion.
Retrospective analyses of the clinical scenarios often
criticise the employment of blood transfusion as ‘too little,
too late’. Women at high risk of losing greater than 1000 mL
should be strongly advised to deliver in a setting where blood
transfusion and intensive care facilities are available.
Blood transfusion may be a life-saving procedure but it is
not without risk. Recipients may rarely develop transfusion-
transmitted infection as well as suffer immunological
sequelae such as red cell alloimmunization.
The major risk of blood transfusion, however, is of a patient
receiving an ‘incorrect blood component’.
Strict adherence to correct sampling, crossmatch and
administration procedures is therefore of paramount
importance, even in an emergency.
Massive blood loss may be defined as:
• Loss of one blood volume within a 24-hour period.
(7% of lean body weight (5 liters in an adult).
27
• Loss of 50% of blood volume within 3 hours.
• Loss of blood at a rate in excess of 150 mL per minute.
Causes of Massive Blood Loss
Obstetric haemorrhage may occur before or after delivery,
but more than 80% of cases occur postpartum.
Worldwide, massive obstetric hemorrhage, resulting from
the failure of normal obstetrical, surgical and/or systemic
hemostasis, is responsible for 25% of the estimated 358,000
maternal deaths each year.2
Blood loss may be:
• Antepartum: Hemorrhage after 24th week of gestation and
before delivery
− Placenta previa, placental abruption, bleeding from
vaginal or cervical lesions
• Postpartum (primary): Within 24 hours of delivery
− Tone (uterine atony)
− Tissue (retained products)
− Trauma (cervical and genital tract damage during
delivery)
− Thrombin (coagulation disorder)
• Postpartum (secondary): 24 hours to 6 weeks post-delivery
− Uterine atony, retained products, genital tract trauma,
uterine inversion.
Blood loss can be notoriously difficult to assess in
obstetric bleeds. Bleeding may sometimes be concealed and
the presence of amniotic fluid makes accurate estimation
challenging.3
Post-partum hemorrhage (PPH) has been defined as a
blood loss of 500 mL or more during puerperium and severe
PPH as a blood loss of 1000 mL or more.4
Goals in the Management of Transfusion
in Severe Hemorrhage Include
• Rapid resuscitation with crystalloids to restore and
maintain the circulating blood volume to prevent tissue
and organ hypoperfusion.
 Chapter 27  Massive Transfusion in Obstetrical Hemorrhage  103
• Maintenance of tissue oxygenation using red blood cells
and
• Reversal or prevention of coagulopathy using appropriate
blood and plasma components, i.e. platelets, fresh frozen
plasma (FFP), for the provision of clotting factors and
cryoprecipitate or fibrinogen concentrate as a source of
fibrinogen.
Prevention and treatment of hypothermia, acidosis and
hypocalcemia will ensure optimal function of transfused
coagulation factors.2
Obstetric conditions associated with the need for blood
transfusion may lead to morbidity and mortality if not
managed correctly. The increasingly important issues in blood
transfusion are adverse events associated with transfusion,
including potential infection and potential transmission
of prions, rising costs and the possible future problems of
availability. Unfortunately, information from randomized
controlled trials to inform best practice is largely unavailable
in the discipline of blood transfusion.
The aim of this guideline is to offer guidance about the
appropriate use of blood products that neither compromises
the affected woman nor exposes her to unnecessary risk.
Strategies to maximize the hemoglobin (Hb) level at delivery
as well as to minimize blood loss are also discussed. It is
envisaged that this guideline will be incorporated into a
protocol for each unit to disseminate locally.
Aim for and Maintain
• Temperature >35°C (pre-warm resuscitation fluids using
temperature controlled blood warmers and use forced air
warming blanket).
• pH >7.2
• Base excess < -6
• Lactate < 4 mmol/L
• Ca2+ >1.1 mmol/L
• Platelets > 50 × 109/L
• PT/APTT <1.5 × normal
• INR ≤1.5
• Fibrinogen >1.5 g/L
It is optimal to have a single person coordinating commu-
nication with the transfusion service, laboratory staff and the
hematologist and/or MedSTAR.
• Phone the transfusion service and identify themselves
(and how to be contacted) as the clinician making the
notification.
• Provide the name, unit record (UR) number and location
of the patient.
• Inform the Transfusion Scientist “Massive Transfusion
Protocol needs to be activated”
• Arrange appropriate care post-event, e.g. care in HDU,
adult ICU or inter-hospital transfer.
Blood Component Therapy
• Historically, aggressive fluid resuscitation with crystalloid/
colloid is recommended to maintain normal blood
pressure but they are complicated by pulmonary edema,
exacerbation of thrombocytopenia and coagulopathy due
to hemodilution.
• On the other hand, permissive hypotension, where systolic
blood pressure of 80–100 mm Hg is tolerated and have
shown survival benefit in some studies.
• It is contraindicated in patients with traumatic head
injuries.
Packed Red Blood Cells (PRBCs)
The majority of protocols recommended six units of PRBCs
be prepared and available and hematocrit be maintained
minimally at 21–24%.1,6,7,9,11-13 These recommendations are
consistent with a recent survey of obstetricians and practice
guidelines from the American Society of Anesthesiologists.14
Ideally, the use of a single unit of PRBCs should increase
hematocrit by approximately 3–4% in a 70 kg patient.15
However, the expected increase in hematocrit may be slightly
less due to expanded blood volume during pregnancy. As
noted elsewhere in this toolkit, any patient with continued
bleeding after initial measures have failed (Stage 2), should
have two units of PRBCs released from the blood bank. If
these are not readily available, consideration should be given
for the use of uncross–matched O negative blood while the
blood bank is completing the patient’s type and cross match.
Consistent with recommendations from this toolkit, for
any patient that reaches Stage 3, a massive OB hemorrhage
pack should be prepared, which includes an additional 4
or 6 units of PRBCs as pre-arranges with the blood bank for
massive transfusion protocol. Good communication with the
laboratory regarding the urgency of the situation is essential.
Fresh Frozen Plasma
Fresh frozen plasma (FFP) contains nearly all coagulation
factors and can be used up to 24 hours after thawing and up
to 5 days if relabeled as “thawed plasma.” Concomitant use of
FFP and PRBCs is recommended during massive hemorrhage.
Using a high ratio of PRBCs to FFP (1.5:1 or 1:1) has been
shown to significantly improve survival from hemorrhage after
trauma.16 Similar recommendations have been established at
centers with existing massive OB hemorrhage protocols with
the goal of maintaining the INR at <1.5–1.7. 1,6,11,17,18 If diffuse
bleeding is noted, or there is laboratory evidence of DIC and
the patient has not been crossmatched, initial requests for 4
units of AB-FFP are recommended. AB plasma is usually in
short supply and reserved for infants. However, 2–3 units of
mismatched plasma can be transfused to adults while type
and cross matching is completed. FFP usually requires 30
minutes to thaw and will not be available immediately. Pre-
arrangement with the blood bank for an inventory of thawed
plasma for immediate issue may be considered if plasma
usage volume is sufficient. If the patient enters into Stage 3
status, there should be no delays in preparation of FFP while
waiting for laboratory results.
104  Section 1  Clinical Hemotherapy

Platelets on maintaining a fibrinogen concentration above 100 mg/

All protocols in our review recommended transfusion of
a single donor apheresis unit when platelet levels varied
between 50 × 109/L and 100 × 109/L.6,8,10,11 Plateletpheresis
units are the standard equivalent of 6 units whole blood-
derived pooled platelets and may increase the platelet count
in a 70 kg patient by approximately 40–50 × 109/L.15 In the
face of massive maternal hemorrhage, platelet transfusions
should maintain platelet count between 50 × 109/L and 100 ×
109/L. However, platelet counts should be used only as a guide
and should be interpreted in conjunction with the patient’s
clinical condition. These recommendations are consistent
with those of the American Society of Anesthesiologists Task
Force on Perioperative Blood Loss.14 Some protocols have
suggested higher platelet counts for initiating transfusion and
maintaining appropriate platelet levels. These suggestions
are based on the assumption that unless bleeding and DIC
have been controlled, the patient will experience ongoing
platelet loss.5,6 Platelets do not require crossmatching and are
generally not type specific. Rh negative platelets are given to
patients with an Rh negative blood type because of the slight
risk of sensitization to the D-antigen.
However, a dose of Rh-immune globulin can be given and
is protective if Rh negative platelets are unavailable.
Cryoprecipitate and Fibrinogen
In the face of hypofibrinoginemia (fibrinogen levels <100–125
mg/dL and ongoing bleeding), fibrinogen should be used in
addition to FFP. Transfusion recommendations were based
dL. Cryoprecipitate release from the blood bank is usually in
groups of 6–10 units. Each unit provides ≥150 mg of fibrinogen
for a total of at least 1500 mg in a pool of 10 units in a total
volume of approximately 80–100 cc. A pooled “ten unit”
pack would be expected to increase the fibrinogen level of a
70 kg patient by approximately 75 mg/dL. It is worth noting
that a 10-unit pool represents 10 separate donor exposures.
If continued bleeding and hypofibrinogenemia is present,
additional units of cryoprecipitate should be used.
Recombinant Factor VIIA
Factor VII is a vitamin K-dependent serine protease with a
pivotal role in coagulation. After reconstitution with sterile
water, each vial contains approximately 0.6 mg/mL (600 µg/
mL). It is marketed for use in patients with hemophilia A
and B. The role of rVII in primary postpartum hemorrhage
is controversial.19,20 It has been reported to significantly
improve hemostasis in hemorrhaging obstetrical patients,
but may also result in life-threatening thrombosis.12 When
avail­able, its use should be reserved for rescue therapy when
conventional therapy has failed (i.e. after 10–12 units of
PRBC, 6–10 units of FFP and 2–3 units of platelets). Dosing
recommendations in obstetrical hemorrhage patients has not
been uniform.
Adverse Effects of Massive
Transfusion
See Table 1.

Table 1: Adverse reactions of transfusions

Type of reaction
Acute adverse effects of transfusion (Onset within minutes or hours)
Hemolysis—Immunologic
(Acute hemolytic transfusion
reaction)
Hemolysis—Physical or
chemical
Febrile nonhemolytic
Anaphylaxis
Urticarial
Transfusion related acute lung
injury (TRALI, non-cardiogenic
pulmonary edema)
Congestive heart failure
Septic complication
Incidence Usual cause Signs or symptoms
1:25,000 Red cell incompatibility, usually ABO Fever, chills, renal failure, DIC, pain,
hypotension, tachycardia, anxiety,
hemoglobinuria, cardiac arrest.
Unknown Overheating, freezing, addition of Asymptomatic hemoglobinuria, rarely
hemolytic drugs or solutions. DIC, renal failure, hypotension
0.5–1.5% Recipient antibodies to donor leukocytes; Fever, chills
or preformed cytokines in blood product
1:20,000–47,000 IgA deficient recipient with antibodies to Respiratory obstruction and
IgA in donor plasma; antibodies to other cardiovascular collapse, angioedema,
plasma proteins, WBCs and platelets anxiety, chills, agitation
1–3% Antibody to donor plasma proteins Pruritis and hives
Reported Donor antibody to recipient leukocytes or Respiratory distress, pulmonary edema
0.001%, patient antibody to donor specific HLA or and hypoxemia with normal wedge
0.02%, granulocytes pressures. “White out” on CXR
0.34%
Unknown Volume overload Respiratory distress
1:1000- Bacterial contamination Usually gram-negative sepsis when the
7:1000- transfusion is red cells, gram-positive
cocci are most common in platelet
transfusion
Contd...
 Chapter 27  Massive Transfusion in Obstetrical Hemorrhage  105

Contd...

Type of reaction Incidence Usual cause Signs or symptoms

Hypothermia Unknown Rapid infusion of cold blood Chills without fever
Hyperkalemia Unknown Rapid infusion of stored red cell Cardiac dysfunction (usually
problematic only in infants or those
with compromised renal function)
Hypocalcemia Unknown Rapid and massive Cardiac dysfunction (usually
Transfusion of stored blood problematic only in patients with
Prophylactic administration of calcium is severe hepatic insufficiency or neonatal
not recommended. massive exchange transfusion)
Delayed adverse effects of transfusion (Onset within days to years)
IMMUNOLOGIC
Delayed hemolytic 1:4000–7000 Alloantibody to RBC antigen, usually Fever, chills, jaundice, pain,
Transfusion reaction anamnestic uncommonly renal failure days to weeks
following transfusion
Graft vs host disease Unknown but Lymphocytes from blood donor mount an Fever, rash, anorexia, diarrhea, LFTs.
rare immune response to host antigens, usually profound pancytopenia which leads to
in an immunocompromised host death
Post-transfusion purpura Rare Alloantibody to platelet antigen (usually Thrombocytopenia and generalized
anti-HPA-1a) purpura
Red cell alloimmunization 2% of transfused Exposure to foregin red cell antigens May cause delayed hemolytic reactions
patients on subsequent transfusions
Platelet-refractoriness 30% of patients Exposure to foreign HLA antigens, Poor response to platelet transfusions
requiring sepsis, depressed hematopoiesis, splenic
multiple plt txs sequestration
Immunomodulation Unknown Leukocytes in transfused products May increase risk of infection or tumor
recurrence
NONIMMUNOLOGIC
Iron overload Dependent on Iron in transfused red cells, usually need Hemochromatosis, cardiac dysfunction
number of red 60+ units in an adult patient
cell transfusion
Delayed adverse effects of transfusion (Onset within days to years)
INFECTIOUS
HIV 1:2,135,000
Hepatitis B 1:205,000
Hepatitis C 1:1,935,000
HTLV I/II 1:2,993,000
CMV < 1% of
seropositive
units
transmit disease
Protozoal infections Rare
(Malaria, Badesia, Chagas
disease)
Parvovirus B19 40–60% of A non-enveloped ssDNA virus which is not Intrauterine infection: May lead to
donors are inactivated by solvent-detergent methods hydrops fetails and fetal demise,
seropositive but of viral inactivation. Has been detected in Children: Fifth’s disease, patients with
viremia occurs pooled factor concentrate products chronic hemolytic syndromes or
only during Immune deficiency: Aplastic crisis
acute phase of
infection
Contd...
106  Section 1  Clinical Hemotherapy
Contd...

Type of reaction Incidence Usual cause Signs or symptoms

Potential or Theoretical Risks
Creutzfelt-Jacob Disease Cases of Abnormal prion which behaves as an Progressive dementia resulting in death
(Theoretical risk) transmission infectious particle
by transfusion
products have
never been
reported or
suspected.
Injection of
buffy coats from
CJD pts directly
into brains of
lab animals
has resulted
in spongiform
encephalopathy
As yet unknown infections Unknown Infectious agents which may be detected in Unknown morbidity and mortality
(Potential risk) the future

SUMMARY 2. It will be the responsibility of the anesthesia team to

maintain the above documentation in the OR (as noted in
During obstetrical hemorrhage, the primary goals are to the PACU or preoperative area.
provide adequate blood product replacement and to either 3. All blood transfusion slips will be filed and attached to the
prevent or correct DIC. The literature and protocols reviewed chart of the patient by the RN/anesthesia.
provided remarkable consensus related to therapy in the
setting of massive obstetrical hemorrhage.
References
1. Bonnar J. Best Practice and Research. Bailliere's Clinical Obstet
Transfusion of Products Gynaecol. 2000;14:1-18.
1. All transfusion, except platelets, will be performed using 2. Mclintock C, James, AH. Obstetric Hemorrhage. Journal of
the level 1 Fluid Warmer/Infuser thrombosis and haemostyasis. 2011;9:1441-51.
3. Davies K, Rucklidge M. Management of Obstetric Haemorrhage.
2. Transfusion will proceed in the following order:
Anaesthesia UK, 2007.
a. 2 units PRBC 4. Royal Australian and New Zealand College of Obstetricians and
b. 2 units FFP Gynaecologists. Management of Postpartum Haemorrhage
c. 5 units pooled platelets (C-Obs 43). Australia, 2011.
d. 2–3 units PRBC 5. Santoso J, Saunders B, Grosshart K. Massive blood loss and
e. 2 units PRBC transfusion in obstetrics and gynecology. Obstet Gynecol Surv.
f. Repeat Steps a-e 2005;60:827-37.
g. 10 Pooled cryoprecipitate 6. University of Washington. Obstetric Emergency Hemorrhage
h. Repeat steps f and g as frequently as deemed necessary Protocol. CMQCC Obstetric, Hemorrhage Toolkit, Obstetric
Hemorrage Care Guidelines and Compendium of Best
based on patient’s status and active blood loss.
Practices. Reviewed by CADPH-MCAH: 11/24/09.
7. Stanford University. Obstetrical Emergency Hemorrhage
Documentation Protocol. CMQCC Obstetric, Hemorrhage Toolkit, Obstetric
Hemorrage Care Guidelines and Compendium of Best
1. Documentation will be maintained during the execution Practices. Reviewed by CADPH-MCAH: 11/24/09.
of the MTP including the following items: 8. California Pacific Medical Center. Obstetrical Hemorrhage
a. Vital signs Q 10 minutes (Temp, BP, HR, RR) Protocol. CMQCC Obstetric, Hemorrhage Toolkit, Obstetric
b. Pulse oximetry Hemorrage Care Guidelines and Compendium of Best
c. Urine output Practices. Reviewed by CADPH-MCAH: 11/24/09.
d. Products administered 9. Barbeiri R. Lessons from Iraq reach the US Labor and Delivery
Suite. OBG Management 20007;19(7):8-11.
e. Time of administration of products
10. Burtelow M, Riley E, Druzin M, Fontaine M, Viele M,
f. Medications administered Goodnough LT. How we treat: management of life-threatening
g. Time of medication administration primary postpartum hemorrhage with a standardized massive
h. Laboratory studies. transfusion protocol. Transfusion. 2007;47(9):1564-72.
 Chapter 27  Massive Transfusion in Obstetrical Hemorrhage  107
11. University of Southern California. Emergency Obstetrical
Hemorrhage Protocol. Approved and in use in Department of
Obstetrics, Women’s and Children’s Hospital.
12. Alfirevic Z, Elbourne D, Pavord S, Bolte A, Van Geijn H, Mercier
F, et al. Use of recombinant activated factor VII in primary
postpartum hemorrhage: the Northern European registry
2000-2004. Obstet Gynecol. 2007;110(6):1270-8.
13. ACOG. Clinical Management Guidelines for Obstetrician-
Gyne­co­­logists. Obstetrics and Gynecology. 2006;108(4):1039-
47.
14. Matot I, Einav S, Goodman S, Zeldin A, Weissman C, U E. A
Survey of physicians' attitudes toward blood transfusion in
patients undergoing cesarean section. American Journal of
Obstetrics and Gynecology. 2004;190(2):462-7.
15. Puget Sound Blood Center. Available at http://www.psbc.org/
therapy/index.htm.
16. Borgman M, Spinella P, Perkins J, Grathwohl K, Repine T,
Beekley A, et al. The Ratio of Blood Products Transfused
Affects Mortality in Patients Receiving Massive Transfusions
at a Combat Support Hospital. The Journal of Trauma.
2007;63(4):805-13.
17. Stinger H, Spinella P, Perkins J, Grathwohl K, Salinas J, Martini
W, et al. The Ratio of Fibrinogen to Red Cells Transfused
Affects Survival in Casualties Receiving MassiveTransfusions
at an Army Combat Support Hospital. The Journal of Trauma.
2008;64(2):S79-S85.
18. Riskin D, et al. Reduced Mortality After Implementation of
a Massive Transfusion Protocol: A Single Trauma Center
Experience. American College of Surgeons. 2008. pp. 13-6.
19. Ahonen J, Jokela R, Korttila K. An open non-randomized
study of recombinant activated factor VII in major postpartum
haemorrhage. Acta Anaesthesiol Scand. 2007;51(7):929-36.
20. Bhuskute N, Kritzinger S, Dakin M. Recombinant factor
VIIa in massive obstetric haemorrhage. Eur J Anaesthesiol.
2008;25(3):250-1.
 Platelet Therapy

Inherited and Acquired

Platelet Disorders:
Diagnosis and Therapy
R Krishnamoorthy
Introduction
Platelets play a central role in hemostasis. Platelets are
produced from megakaryocytes in the bone marrow and have
a life span of 8 to 10 days. Normal platelet count is 150–450 ×
109/L; less than 150 × 109/L is thrombocytopenia and more
than 450 × 109/L is thrombocytosis. Structure: Platelets are
2–3 microns in size and do not have a nucleus. Platelets have
dense granules (contain ATP, ADP), alpha granules (contain
platelet specific proteins like Platelet Factor 4 and Growth
factors like PDGF) and lysosomal granules (contain elastases
and collagenases). Platelets also have an open canalicular
system through which the content of the platelet granules are
extruded during platelet secretion and aggregation. Platelets1
have glycoprotein receptors—important among them are
GP Ib-IX-V complex (binds to vWF and is responsible for
adhesion) and GP IIb-IIIa complex (binds to fibrinogen and
is responsible for aggregation). Role of platelets in hemostasis
are adhesion to the sub endothelial collagen (via vWF) at sites
of vascular injury followed by activation and aggregation to
form a primary hemostatic plug.
28
• Electron microscopy helps in assessment of platelet
granule defects and changes in platelet ultrastructure.
Discussion
Platelet disorders encompass a variety of Hereditary and
Acquired diseases that can be divided in to:
1. Quantitative: Platelet disorders3 (disorders of platelet
number): Thrombocytopenia and thrombocytosis.
2. Qualitative: Platelet disorders (disorders of platelet
function).
Some overlap exists between these two major categories.
Thrombocytopenia4 may be caused by:
• Decreased production of platelets (due to primary bone
marrow disorders, bone marrow invasion, bone marrow
injury, nutritional disorders, hereditary throm­bocytopenic
syndromes).
• Increased destruction5 of platelets as given in Flow chart 1.
Flow chart 1  Causes of destruction of platelets

Methodology
Patient’s clinical history and family history of bleeding are
very important. Laboratory investigations carried out in case
of platelet disorders are:
• Platelet count and peripheral blood smear
• PT, APTT, TT to rule out coagulation disorders
• Bleeding time has gradually fallen out of favor as a
clinically useful test. But a prolonged bleeding time should
be considered sufficient to perform additional tests.
• Platelet function analyzer (PFA-100)2
• Platelet aggregation studies using agonists such as ADP,
thrombin, epinephrine, ristocetin.
• Measurement of platelet ADP and ATP content and release
are useful in diagnosis of storage pool and release defects.
• Flow cytometry can be used to measure surface receptor
density in BSS, GT.
 Chapter 28  Inherited and Acquired Platelet Disorders: Diagnosis and Therapy  109
• Sequestration as in hypersplenism.
Thrombocytosis may be primary (e.g. polycythemia vera,
MDS) or secondary (e.g. iron deficiency, malignancy).
Qualitative platelet disorders6 may be:
• Congenital:
– Disorders of adhesion—Bernard Soulier syndrome7
(BSS) (defect in GP Ib-IX) is a rare autosomal recessive
disorder. Giant platelets8 are seen on peripheral blood
smear in BSS.
In Von Willebrand disease (vWD), vWF binding to GP
Ib-IX-V is reduced.
– Disorders of activation—storage pool deficiency
– Disorders of aggregation—Afibrinogenemia, Glanz­
mann’s Thrombasthenia (GT) (defect in GP IIb-IIIa) is
a rare autosomal recessive entity.
– Disorder of platelet procoagulant activity – Platelet
Factor V Quebec
• Acquired (more common):
– Uremia
– Myeloproliferative disorders like polycythemia vera,
myelofibrosis and essential thrombocytosis
– MDS and acute leukemias are clonal disorders in which
all cell lines are abnormal
– Dysproteinemias-paraproteins may coat platelets non-
specifically and interfere with binding
– Cardiopulmonary bypass is associated with platelet
dysfunction
– Antiplatelet antibodies
– Liver disease, various drugs (e.g.) aspirin, heparin.
Clinical Features of Platelet Disorders
Bleeding occurs with falling platelet counts. Sites of bleeding
may be Cutaneous (petechiae, purpura), Mucosal (epistaxis,
menorrhagia, GIT bleed) or within the central nervous system.
In qualitative platelet disorders, count is usually normal
but bleeding time (BT) is prolonged due to abnormalities
in function. Clinical features vary. Most patients have
mucocutaneous bleeding.
Diagnosis
Bedside examination, accurate patient and family history is a
key element in assessing platelet disorders.
Management
Bernard Soulier syndrome: General and specific treatment
of bleeding, contraceptive therapy after puberty in females,
Platelet transfusion (preferably HLA matched) to treat
bleeding during surgery, Desmopressin and rFVIIa. Rarely
hematopoietic stem cell transplant (HSCT).
Glanzmann’s thrombasthenia9: Prophylactic platelet trans­
fusion, Fibrin sealants and antifibrinolytics like Tranexamic
acid to treat localized bleeding, high doses of Progesterone to
treat menorrhagia; rFVIIa may help arrest bleeding. BMT and
gene therapy have been tried.
Uremia: Hemodialysis/peritoneal dialysis; RBC transfusion
and EPO have been observed to decrease bleeding and
increase platelet adhesiveness but some patients become
hypercoagulable.
Platelet Transfusion Triggers10
Prophylactic: Maintain platelet count in all patients > 10 ×
109/L or in stable patients > 5 × 109/L and in patients with
coagulopathy > 20 × 109/L. In patients who are already
bleeding or who are about to undergo a hemostatic challenge
such as a surgical procedure, attempts are made to keep the
platelet count > 50 × 109/L. An even higher target of 100 × 109/L
is often used for intracerebral, pulmonary and ophthalmic
hemorrhage. The role of anemia should also be considered in
prevention or treatment of hemorrhage.
Therapeutic dose of platelets is 1 unit of RDP/10 kg body
weight or 1 unit of SDP in adults. Calculating the corrected
count increment (CCI) and percent platelet recovery (PPR)
will help in determining the adequacy of response to platelet
transfusion.
Conclusion
Understanding the basic roles of platelets, bone marrow,
spleen and immune system in normal conditions and specific
diseases will aid in determining the cause of an abnormal
platelet count. A fundamental knowledge of mechanisms
involved in platelet adhesion and aggregation allows for
better understanding of platelet functional disorders.
References
1. Buchanan GR. Quamtitative and Qualitative platelet disorders.
Clin Lab Med. 1999;19:71-86.
2. Cines DB, Blanchette VS. Immune thrombocytopenia purpura.
N Engl J Med. 2002;346:995-1008.
3. Drews RE, Weinberger SE. Thrombocytopenic disporders in
critically ill patients. Am J Respir Crit Care Med. 2000;162:347-
51.
4. Fausett B, Silver RM. Congenital disorders of platelet function.
Clin Obstet Gynecol. 1999;42:390-405.
5. George JN. Platelets. Lancet. 2000;355:1531-9.
6. Lankford KV, Hillyer CD. Thrombotic thrombocytopenic
purpura: new insights in disease pathogenesis and therapy.
Transfus Med Revs. 2000;12:244-57.
7. Mhawech P, Saleem A. Inherited giant platelet disorders. Am J
Clin Pathol. 2000;113:176-90.
8. Rodgers GM. Overview of platelet physiology and laboratory
evaluation of platelet function. Clin Obstes Gynecol. 1999;
42:349-59.
9. Shapiro AD. Platelet function disorders. Haemophilia. 2000;
6(Suppl 1):120-7.
10. Tullu MS, Dixit PS, Nair SB, et al. Glanzmann’s thrombasthenia.
Indian J Pediatr. 2001;68:563-6.
Transfusion Practice in Dengue
BL Bhardwaj, Harnoor Singh Bhardwaj
Dengue is the most prevalent mosquito borne viral disease
and has become a global public health concern affecting 50–
100 million people every year. South–Asian countries such
as India, Indonesia, Myanmar and Thailand are at a highest
risk of dengue accounting for nearly half of the global risk.1
WHO has classified dengue into dengue fever (DF), dengue
hemorrhagic fever (DHF) and dengue shock syndrome
(DSS).2 DF is defined clinically as the acute febrile illness with
two or more manifestations (headache, retro-orbital pain,
myalgia, arthralgia, rash, hemorrhagic manifestations or
leucopenia) and occurring at the same location and time as
other confirmed cases of DF.
DHF is characterized as increased vascular permeability
that leads to a capillary leak syndrome. It must meet the
following 4 criteria: (i) Fever or history of fever lasting 2–7
days, (ii) Hemorrhagic tendency, shown by positive tourniquet
test or spontaneous bleeding, (iii) Thrombocytopenia
with platelet count ≤ 100 × 109/L, (iv) Evidence of plasma
leakage shown either by hemoconcentration with sub­
stantial changes in serial measurements of packed cell
volume or by development of pleural effusion, ascites or
both. The thrombocytopenia develops 5–7 days after the
dengue infection and probably coincides with the antibody
production against dengue virus full length nonstructural
protein 1 (NS1). This antibody cross react with human
platelets and inhibit platelet aggregation leading to platelet
dysfunction and bleeding. There is shift of intravascular
to interstitial and serosal compartment.3 DHF is further
classified into four severity grades according to the presence
of spontaneous bleeding and the severity of plasma leakage
(Table 1). DSS refers to DHF grade III and IV in which shock is
present along with four DHS defining criteria. Moderate shock
identified by narrowing of the pulse pressure or hypotension
for age, is present in grade III DHF, whereas profound shock
with no recordable pulse or blood pressure is present in
grade IV. The presence of thrombocytopenia with concurrent
hemoconcentration differentiates Grade I and Grade II DHF
from DF. The bleeding is one of the dreaded complications of
DHF/DSS. It is variable and does not always correlate with the
29
laboratory abnormalities in the coagulation profile. Bleeding
in DF/DHF/DSS results from a combination of factors such
as thrombocytopenia, coagulopathy and vasculopathy.1
Diagnosis of dengue is by serological assays such as IgM
and IgG ELISA, a week after infection. For confirmation RT
PCR assays are done. Before platelet transfusion is given to
these patients, coagulation profile should be done to rule out
the cause of bleeding. Platelet transfusion is given in those
patients who are either bleeding or having other hemorrhagic
symptoms along with thrombocytopenia.4
CLINICAL MANAGEMENT
As per the guidelines of the Directorate of National Vector
Borne Diseases Control Program, Director General of Health
Services, Government of India5 regarding clinical manage­
ment of DF, DHF and DSS management, blood transfusion is
recommended in cases with unstable vitals where hematocrit
falls at the rate of 10 mL/Kg/hr (Flow charts 1 and 2).
Indications of Red Cell Transfusion
• Loss of blood—10% or more of total blood volume.
Preferably fresh whole blood/components to be used.
• Refractory shock despite adequate fluid administration
and declining hematocrit.
Replacement volume should be 10 mL/Kg body wt at a
time and coagulogram should be done. If fluid overload is
present packed red cells are to be given.
Relationship of Platelet Counts and
Hemorrhage in Dengue Fever
The mechanisms of hemorrhage in dengue are multifactorial
and incompletely understood.6 The presence or degree of
thrombocytopenia alone is not associated with increased
bleeding risks in dengue. In DSS,7 only modest increase in
prothrombin time (PT) and partial thromboplastin time
(PTT) was noted. Plasma concentration of anticoagulation
 Chapter 29  Transfusion Practice in Dengue  111

Table 1: Grading the severity of dengue infection5

DF/DHF Grade Symptoms/signs Laboratory findings
DF Fever with two or more of following: Leukopenia, thrombocytopenia
- Headache
- Retro-orbital pain
- Myalgia
- Arthralgia
DHF I Above criteria for DF plus positive Thrombocytopenia: Platelet count less than
tourniquet test, evidence of plasma leakage 100,000/cumm
Hematocrit rise 20% or more
DHF II Above signs and symptoms plus Thrombocytopenia platelet count less than
some evidence of spontaneous bleeding in skin or other organs (black 100,000/cumm
tarry stools, epistaxis, bleeding from gums, etc.) Hematocrit rise 20% or more
and abdominal pain
DHF III Above signs and symptoms plus circulating failure (weak rapid pulse, Thrombocytopenia: Platelet
pulse pressure <20 mm Hg or high diastolic pressure, hypotension with count less than 100,000/cumm
the presence of cold clammy skin and restlessness) Hematocrit rise more than 20%
DHF IV Profound shock with undetectable blood pressure or pulse Thrombocytopenia: Platelet count less than
100,000/cumm
Hemotocrit rise more than 20%
Note:
•  DF may sometimes present with bleeding manifestation but without evidence of hemaconcentration/plasma leakage. It should not be confused
with DHF.
•  In addition pain and/or tenderness in upper abdomen have been commonly observed as prominent clinical feature.

proteins C, S and antithrombin III were noted to decrease
with increasing severity of shock, likely due to increased
capillary leakage. Increased thrombomodulin, tissue factor
and plasmin activator inhibitor type-1 (PAI-1) were observed
with thrombomodulin levels increasing with severity of shock,
and PAI-1 levels increasing with severity of bleeding. Direct
activation of fibrinolysis by dengue virus has been implicated,
a process that distinguishes hemorrhage in dengue from that
seen in classic disseminated intravascular coagulopathy.
The presence of fever is viewed as an additional risk factor
for bleeding in the presence of severe thrombocytopenia.
Severe thrombocytopenia in dengue occurs around the tail
end of the febrile period. There is no significant relationship
between bleeding severity and degree of thrombocytopenia.3
Prophylactic Platelet Transfusions in Dengue
Quantifiable increases in platelet counts and other coagulation
parameters are transient in dengue, even more so than in
other conditions where prophylactic platelet transfusions are
administered. In DSS patients who underwent prophylactic
platelet transfusion, platelet counts, prothrombin time ratio
(PTR), and partial prothrombin time (PTT) returned to pre-
transfusion values in less than 5 hours, demonstrating the
lack of clinical efficacy as an intervention which happens
to target a parameter—platelet counts—which is not the
implicated culprit of major bleeding in dengue.8
The British Committee for Standards in Hematology
(BCSH)9 states that the threshold of 10 × 109/L is as safe as
the higher levels for patients without additional bleeding
risk factors such as sepsis, concurrent use of antibiotics or
other abnormalities of homeostasis, prolonged high fever (as
seen in dengue patients with severe hepatitis and secondary
bacterial infections), sepsis, vascular compromise, anatomic
aberrations, significant coagulopathy, uncorrected hemo­
dynamic instability, DHF and severe dengue. Further as
per BCSH guidelines,9 a platelet transfusion threshold of
5 × 109/L is identified for patients without any bleeding risk
factors if alloimmunization leading to platelet refractoriness
is a concern. Annals Academy of Medicine Singapore3
propose that prophylactic platelet transfusion in dengue
patients may be safely withheld for platelet counts as low as
5 × 109/L in the absence of additional bleeding risk factors
and should be reserved for patients with major bleeding
(Grade 2 and above in WHO bleeding scale). Careful
observation without platelet transfusion is warranted in
stable dengue patients with only minor ‘dry’ bleeding such
as petechiae, ecchymosis and minor ‘wet’ bleeding such as
mucosal oozing (WHO bleeding scale Grade 1). Singapore’s
clinical practice guideline recommends that in the presence
of additional bleeding risk factors or when there is rapid
decline in platelet count, prophylactic platelet transfusion
in pts < 20 × 109/L should be given.10 As per the guidelines
of the Directorate of National Vector Borne Diseases Control
Program, Director General Of Health Services, Govt of India5
guidelines for dengue treatment, platelet transfusion should
be given at platelet count < 10 × 109/L without additional risk
factors.
112  Section 1  Clinical Hemotherapy
Flow chart 1  Volume replacement flow chart for patients with DHF Grades I and II5

Improvement : Hematocrit falls, pulse rate and blood pressure stable, urine output rises.
No improvement : Hematocrit or pulse rate rises, pulse pressure falls below 20 mm Hg, urine output falls.
Unstable vital signs : Urine output falls, signs of shock.

Platelet Transfusion • Platelet count <10 × 109/L in absence of bleeding manifes-

Platelet concentrates (PCs) prepared from whole blood dona­
tions or plateletpheresis single donor platelets (SDP) are used.
Their use varies. The whole blood derived platelets random
donor platelets (RDP) are either pooled PCs or unpooled PCs.
The efficacy of these preparations is dependent on multiple
factors including residual leukocyte count, plasma content
and additive solution, etc.11
Indications of Platelet Transfusion5 (Table 2)
In general there is no need to give prophylactic platelets even at
< 20 × 109/L. Prophylactic platelet transfusion may be given at:
tations.
• Prolonged shock with coagulopathy and abnormal
coagulogram.
• In case of systemic massive bleeding, platelet transfusion
may be needed in addition to red cell transfusion.
Dose of Platelets
Therapeutic dose of platelet depends on the patient’s
pre-transfusion platelet count and clinical condition. The
practice of transfusing random donor platelets at a dose
of 1 unit per 10 Kg body weight or of transfusing 1 unit
of apheresis platelets from one donor is common. The
 Chapter 29  Transfusion Practice in Dengue  113

Flow chart 2  Volume replacement flow chart for patients with DHF Grades III and IV 5

•  Serial platelet and hematocrit determinations:—Drop in platelets and rise in hematocrit are essential for early diagnosis of DHF.
•  Cases of DHF should be observed every hour for vital signs and urinary output.

Table 2: Criteria for platelet transfusion in dengue hemorrhagic fever5

Indications Prophylactic transfusion Pl count < 10 × 109/L without additional risk factors
Therapeutic transfusion •  Significant active bleeding with platelet count < 50 × 109/L
•  Proven disseminated intravascular coagulation
•  Prolonged shock with coagulopathy and abnormal coagulogram
•  In systemic massive bleeding, Pl transfusion may be needed in addition to red cell transfusion
Dosage Adults 1 units of random donor platelet concentrate/10 Kg Body Wt
Neonates 1 unit of random donor platelet concentrate/ 2.5 Kg Body Wt
Response Clinical response Cessation of bleeding
to platelet Platelet count At 1 hour post transfusion
transfusion increment (PCI)
Courtesy: DGHS, Government of India guidelines/WHO/British Committee for Standards in Hematology (BCSH)
114  Section 1  Clinical Hemotherapy
Table 3: Platelet increments after one unit of random donor
platelet transfusion5
In adult : 5–10 × 109/L in an adult of 70 Kg body weight
In child : 20 × 109/L in child of 18 Kg body weight
In infant : 75–100 × 109/L
effect of a transfusion is judged by the clinical results and
the platelet count obtained one hour later. Table 3 shows
platelet increment from each unit of random donor platelets
prepared from 450 mL blood under optimal conditions. One
unit of platelet from apheresis donor will raise the platelet
count of an adult of 70 Kg body weight approximately by 30–
60 × 109/L under optimal conditions. The clinical response to
platelet transfusion is diminished by fever, hypersplenism,
infections, splenomegaly, uremia, medications, concomitant
coagulation disorders, alloimmunization to HLA or platelet
antigens. A standard 170 micron filter is recommended for
the administration of platelets.
Effect of platelet transfusion is monitored by estimating
levels of Bleeding time; Platelet count; aPTT and PT and
Fibrinogen. ABO compatibility between donor and recipient
is of minor importance in platelet transfusion in most
adults. Administration of ABO incompatible platelets is
an acceptable transfusion practice. The possibility of Rh
immunization by red cells contained in platelet concentrate
should be considered in female patients. Rh (D) negative
female patient of child bearing age should be given platelets
from Rh (D) negative platelets, otherwise Rh (D) immune
globulin (RhDIg) should be given in doses of 20 µg for each
unit of platelet.
Each unit of random donor platelets contains 5.5 × 1010
platelets, so that 5-packs contain 2.75 × 1011. Single donor
platelet pack contains 3 × 1011 platelets.
Corrected count increment (CCI) = (platelet increment
per µL) × (body surface area in m2)/number of platelets
transfused (× 1011). The expected CCI 10–60 minutes after a
platelet trans­fusion is 7500 or greater representing 20–30%
platelet recovery.
Platelet refractoriness is defined as a CCI less than 7500 on
at least two sequential platelet transfusions.
A “quick and dirty” method for estimating platelet
refractoriness assumes that the normal increment in platelet
count after transfusion of 1 unit/10 Kg of single donor platelets
is 50 × 109/L at 10–60 min and 40 × 109/L at 18–24 hours. A one
hour count less than 30% of expected, or a 24 hour count less
than 20% of expected, represents an inadequate response to
transfusion.
When immune platelet refractoriness is suspected on the
basis of poor CCI’s, the next steps are to order a screen for anti-
HLA antibodies and, if positive, use HLA-selected platelets.
Nonimmune causes of low CCIs include fever, infection,
bleeding, splenomegaly, gamma-irradiation, DIC, extensive
surgery, and concurrent amphotericin B therapy. Non-
immune platelet consumption is the most likely cause of a
low CCI when no alloantibody can be found by the laboratory
and the patient has been transfused for less than three weeks
(unless there has been prior exposure to HLA antigens by
transfusion, organ transplantation or pregnancy).12
Risks of Platelet Transfusion
Platelet transfusion carries with it a variety of risks including
alloimmunization with resulting refractoriness, allergic
reactions, febrile non-hemolytic reactions; transfusion
associated acute lung injury, viral, bacterial and parasitic
infections.13-16 Platelets carry an additional non-negligible
risk of bacterial contamination due to the fact that its storage
is done at room temperature for up to 5 days compared
with other blood products which are stored in the blood
bank refrigerator or frozen. It is estimated that 1 in 1000 to
3000 platelet units are contaminated with bacteria, resulting
in life-threatening incidents in 1 of 100,000 recipients and
immediate fatal outcomes in 1 of 500,000 recipients.13 The
presence of leukocytes in platelet preparations is largely
responsible for the febrile, nonhemolytic, transfusion
reactions via the generation of cytokines, occurring in 30% to
40% of platelet transfusion recipients. Alloimmunization may
result in refractoriness to platelet transfusion in the future,
when it may be really necessary. Platelet transfusion can
also cause significant hypotension in patient on angiotensin
converting enzyme inhibitors by generation of large amounts
of bradykinin by filtration of platelet concentrates through a
negatively charged filter.3
Early recognition of dengue with prompt correction
of hemodynamic parameters remains the cornerstone of
avoiding hemorrhage and ensuring good clinical outcomes.6
Platelets concentrates due to their short shelf life are
frequently in limited supply. Hence, appropriate use of blood
is required to ensure the availability of blood for patients
in whom it is really indicated, as well to avoid unnecessary
exposure of the patients to the risk of transfusion reactions
and transmission of blood borne infection.
Use of Fresh Frozen Plasma/Cryoprecipitate5
In coagulopathy with bleeding as per advise of physician and
patient’s condition.
Role of Recombinant Activated
Factor VII (rFVIIa)
It is a useful adjunct treatment for controlling active bleeding.
It also decreases the need of platelets.17
These are only broad guidelines. The treating physician
should consider the condition of the patient in totality and
decide.

References
1. Shivbalan S, Anandnathan K, Balasubramanium S, et al.
Predictors of spontaneous bleeding in dengue. Ind Pediatr.
2004;71:33-6.
2. Dengue Guidelines for Diagnosis, Treatment, Prevention and
Control–World Health Organization, 2009. Available at http://
whqlibdoc.who.nt/publications/2009/9789241547871_eng.
pdf. Accessed July 1, 2011.
3. The Rationale Use of Platelet Transfusion in Dengue Fever.
Transfusion Newsletter 8th edn. 2013.pp.1-4. Blood Trans­
fusion Service of Health Bureau Government of Macao SAR,
crystal @ssm. gov. mo.
4. Chairulfatah A, Setiabudi D, Agoes R, Colebunder R.
Thrombocytopenia and platelet transfusion in dengue and
dengue shock syndrome. WHO Dengue Bulletin. 2003;27:141-3.
5. Clinical Management. In: Guidelines for Clinical Management
of Dengue fever, dengue haemorrhagic fever and dengue shock
syndrome. Directorate of National Vector Borne Diseases
Control Program. DGHS Ministry of Health and Family Welfare,
Govt of India. 2008.pp.14-24.
6. Changa K, Dimatatac F, Diana LT Teo, et al. When less is
More: Can We Abandon Prophylactic Platelet Transfu­
sion in Dengue Fever? Ann Acad Med Singapore. 2011;40:
539-45.
7. Wills BA, Oragui EE, Stephens AC, et al. Coagulation abnor­
malities in dengue hemorrhagic Fever: serial investigations in
167 Vietnamese children with Dengue shock syndrome. Clin
Infect Dis. 2002;35:277-85.
Chapter 29  Transfusion Practice in Dengue  115
8. Lum LC, Abdel-Latif Mel-A, Goh AY, Chan PW, Lam SK.
Preventive transfusion in Dengue shock syndrome—is it
necessary? J Pediatr. 2003;143:682-4.
9. Guidelines for the use of platelet transfusions. Br J Haematol.
2003;122:10-23.
10. Health Sciences Authority–Ministry of Health Clinical
Practice Guidelines–Clinical Blood Transfusion, Singapore.
2011;3(5):48.
11. Schrezenmeir H, Seifried E. Buffy-coat–derived pooled platelet
concentrates and apheresis platelet concentrates: which
product should be preferred? Vox Sang. 2010;99(1):1-15.
12. Delaflor-Weiss E, Mintz PD. The evaluation and management
of platelet refractoriness and alloimmunization. Transfus Med
Rev. 2000;14:180-96.
13. Fatal bacterial infections associated with platelet transfusions−
United States, 2004. MMWR Morb Mortal Wkly Rep. 2005;
54:168-70.
14. Epidemiological News Bulletin: Management of guidelines for
dengue patients at Tan Tock Seng Hospital and communicable
Disease center Singapore. Platelet transfusion. 2005. p.49.
15. Dodd RY. Current viral risks of blood and blood products. Ann
Med. 2000;32:469-74.
16. Dwyre DM, Fernando LP, Holland PV. Hepatitis B, hepatitis
C and HIV transfusion − transmitted infections in the 21st
century. Vox Sang. 2011;100:92-8.
17. Chuansumrit A. Control of Bleeding in children with dengue
haemorrhagic fever using recombinant activated factor VII: a
randomized, double blind, placebo-controlled study. Blood
Coagul Fibrinolysis. 2005;16(8):549-55.
Fetal and Neonatal Alloimmune
Thrombocytopenia
Prasun Bhattacharya
Fetal and neonatal alloimmune thrombocytopenia (FNAIT)
is the most common cause of severe thrombocytopenia in the
fetus and otherwise healthy neonates.1 The overall incidence
of this entity in the Caucasian population has been estimated
by prospective studies to be 1 in 800 to 1 in 1000 live birth.2,3
This condition results from incompatibility between the
parents for platelet-specific antigens.
These antigens are inherited in the fetus in an autosomal
co-dominant fashion. Fetomaternal passage of platelets
results the development of the specific antibodies to the
exposed fetal antigen, which is absent in the pregnant mother.
This syndrome may be comparable to the hemolytic disease
of the newborn, although severe thrombocytopenia may
occur during the index pregnancy.4-6 The thrombocytopenia
results from the maternal immunization against specific
platelet alloantigens paternally inherited by the fetus. These
allo-antibodies can cross the placental barrier as early as 14
weeks of gestation.7 The maternal IgG coated fetal platelets
are cleared in the reticuloendothelial system resulting mild to
severe thrombocytopenia. The most significant complication
of FNAIT is intracranial hemorrhage (ICH) which occurs in
10–20% of affected pregnancies, with 75% of them occurring
before birth.8
Pathogenesis of FNAIT
Human platelet antigens (HPA) are specific proteins on
platelet membrane, HPA system is used in the nomen­
clature of platelet antigens. There are 24 platelet-specific
alloantigens, that have been recognized. They are numbered
according to the chronological order of discovery. The
platelet alloantigen is then further defined according to the
frequency in which it appears in the population. A high-
frequency alloantigen is designated ‘‘a’’ and a low-frequency
alloantigen is designated ‘‘b.’’ HPA-1a is the most common
platelet alloantigen in the Caucasian population and is
responsible for 80% of the cases of NAIT. The presence of
antibodies to the HPA-1a antigen is also strongly associated
30
with the HLA DRB 3*0101. The other antigens involved are
HPA–5b, HPA-1b and HPA-15.9
Clinical Presentation
Since its first description by Harrington in 195310, the
diagnosis of NAIT in the first born child is usually unexpected
by the family and health care providers and is frequently made
at birth. The infant can present with petechiae, bruising, and
bleeding but will otherwise look well. The neonate may be
asymptomatic with incidental thrombocytopenia (platelets
count< 150 x 10 9/L).
With the development of fetal medicine, fetal alloimmune
thrombocytopenia has been recognized as the most severe
form of thrombocytopenia. By the end of the 1st trimester
the fetal platelet count reaches the threshold value, i.e. 150 ×
109/L. Fetal alloimmune thrombocytopenia may be suspected
in a case of ICH upto 80% and of them 40% occurring before
30th gestational weeks.11
Laboratory Investigations
When to suspect NAIT in the fetus:
• If fetal thrombocytopenia is incidentally discovered
• If there is a fetal ICH, hydrocephalus or ventriculomegaly
or cerebral cysts
• If there is a family history of NAIT if there is unexplained
fetal anemia
• If there are unexplained, recurrent miscarriages after the
first trimester
Detection of maternal platelet-specific antibodies has
to carried out by two techniques, indirect platelet immuno­
fluorescence test (PIFT) and by monoclonal antibody
specific immobilization of platelet antigens (MAIPA). Platelet
typing of the parent and the newborn may be done by the
molecular techniques as polymerase chain reaction (PCR)
SSP (sequence specific primer) and PCR-RFLP (restriction
fragment length polymorphism).
 Chapter 30  Fetal and Neonatal Alloimmune Thrombocytopenia  117
Therapy and Management
Antenatal Management
Routine screening to identify fetuses at risk for hemolytic
disease of the newborn is well established. A similar screening
program of all pregnant women to identify those fetuses at
risk for NAIT is not routinely performed.
A mother with an identified personal or family history
of NAIT allows health care professionals to provide close
surveillance and early antenatal management during the
pregnancy.
The goal of antenatal therapy is to reduce the severity of
thrombocytopenia and thereby reduce the complications of
ICH, long-term disability, and death. Options for therapy include
in utero transfusions of antigen compatible platelets, intra-
venous immunoglobulin (IVIG), and systemic corticosteroids.
Although majority of these approaches are observational, but
it is universally acceptable that these high-risk mothers require
specialized referral center for management.
Newborns
Infants with clinical bleeding and platelet count below 30 ×
109/L require prompt platelet transfusions.12 The best choice
is to transfuse antigen-negative platelets from donors or
washed and irradiated maternal platelets. The newborn must
be monitored closely until the trigger point has been reached.
In the absence of antigen negative platelets the neonate may
be transfused with random platelet with addition of IVIG at a
dose of 1g/kg of body weight.
Conclusion
The potentially devastating sequelae of alloimmune throm­
bo­cytopenia always need a rapid response from the health
care system in regard to diagnosis and treatment. In the
absence of large scale, randomized controlled trials, there
is hardly standard guidelines among health care providers
regarding the optimum management of FNAIT. The diagnosis
and care of the infant requires a coordinated, collaborative
effort between obstetrics, neonatology, hematology, and
transfusion medicine.
References
1. Israels SJ. Thrombopoietin and neonatal throm­bocytopenia.
Paed Res. 2000;47:176-7.
2. Durand-Zaleski I, Schlegel N, Blum-Boisgard C, Uzan S,
Dreyfus M, Kaplan C. The immune thrombocytopenia working
group. Screening primiparous women and newborns for
fetal/neonatal alloimmune thrombocytopenia: a prospective
comparison of effectiveness and costs. Am J Perinatol.
1996;13:423-31.
3. Williamson LM, Hackett G, Rennie J, Palmer CR, Maciver C,
Hadfield R, et al. Natural history of fetomaternal alloimmuni­
zation to the platelet specific antigen HPA–1 a (PLA1, Zwa) as
determined by antenatal screening. Blood. 1998;92:2280-7.
4. Bussel JB, Berkowitz RL, Lynch L, Lesser ML, Paidas MJ,
Huang CL, et al. Antenatal management of alloimmune
thrombocytopenia with intravenous gamma globulin: a
randomized trial of the addition of low dose steroids to intra-
venous gamma globulin. Am J Obstet Gynecol. 1996;174:
1414-23.
5. Kanhai HH, Porcelijn L, van Zoeren D, Klumper F, Victor
H, Meerman RH, et al. Antenatal care in pregnancies at risk
of alloimmune thrombocytopenia: report of 19 cases in 16
families. Eur J Obstet Gynecol. 1996;68:67-73.
6. Silver RM, Porter TF, Branch DW, Esplin MS, Scott JR. Neonatal
alloimmune thrombocytopenia: antenatal management. Am J
Obstet Gynecol. 2000;182:1233-8.
7. Kaplan C. Neonatal alloimmune thrombocytopenia. Haema­
tologica. 2008;93:805-7.
8. Pearson HA, Shulman NR, marder VJ, Cone TE jr. Isoimmune
neonatal thrombocytopenic purpura: clinical and therapeutic
considerations. Blood. 1964:23;154-77.
9. Symington A, Bosco P. Fetal and neonatal alloimmune
thrombocytopenia: harvesting the evidence to develop a
clinical approach to management. Am J Perinatol. 2011;28(2):
138-44.
10. Harrington WJ, Sprague CC, Minnich V, Moore CV, Aulvin
RC, Dubach R. Immunologic mechanisms in neonatal
and thrombocytopenic purpura. Ann Intern Med. 1953;38:
433-69.
11. Spencer JA, Burrows RF. Feto-maternal alloimmune throm­
bocytopenia: a literature review and statistical analysis. Aust
NZ J Obstet Gynecol. 2001;41:45-55.
12. Bassler D, Greinacher A, Okascharoen C. A systematic review
and survey of the management of unexpected neonatal
alloimmune thrombocytopenia. Transfusion. 2008;48:92-8.
Clinicopathological Syndrome
in Immune-mediated Heparin-
induced Thrombocytopenia
P Arumugam
Heparin-induced thrombocytopenia (HIT) is the most impor­
tant and most frequent drug-induced, immune mediated
type of thrombocytopenia.1 It has an immunologic basis that
is not yet fully understood and, in contrast to other drug-
induced causes of thrombocytopenia, is associated with a
thrombotic rather than a bleeding tendency. This condition
was previously called type II HIT to distinguish it from mild
and clinically benign type I HIT which is sometimes observed
early in the course of heparin administration, is non-immune
mediated and self-limiting, and is not associated with a
bleeding or clotting tendency.
Pathogenesis
Platelet Factor 4 (PF4) is a chemokine produced by megakar­
yocytes and released from platelet α-granules upon platelet
activation. Released PF4 binds cell-surface glycosaminogly­
cans and heparin. Formation of the PF4-heparin complex
causes a conformational change in the protein leading to
exposure of cryptic epitopes to which the antibodies in HIT
are usually generated.2 The anti-PF4/heparin antibodies,
which are of IgG isotype, bind to PF4 through their F(ab)
domains. HIT antibodies are transient with a median time
to disappearance of 50–80 days.3 Serial PF4 molecules align
on the platelet surface and several IgG molecules bind,
leading to the formation of large immune complexes that
cross-link the platelet FcγIIa receptors, and resulting in
platelet aggregation and associated platelet procoagulant
response.4 HIT antibodies also bind to endothelial cells
and monocytes resulting in additional procoagulant effects
through increased expression of tissue factor and formation
of platelet-monocyte aggregates leading to thrombin
generation. Therefore in HIT the clinical manifestations are
the result of both FcγIIa-dependent platelet activation and
activation of the coagulation system through tissue factor
expression and platelet activation.
Pathophysiology of HIT. (1) Heparin binds with PF4 and
acts as immunogens. (2) IgG antibody thus produced forms
31
PF4–heparin–IgG multimolecular complex. (3) The complex
then binds via Fc receptor to platelets and activates them
(4a) activated platelet releases additional PF4 and (4b)
prothrombotic microparticles. (5) Immune complex interacts
with endothelial cells and promotes immune-mediated
endothelial damage (Fig. 1).
Clinical features
The incidence of HIT is up to 4% in patients treated with
unfractionated heparin. Low-molecular-weight heparin is
less likely to be associated with HIT.5,6 Bovine heparin appears
somewhat more likely to cause the syndrome than porcine
heparin.6 In HIT, a reduction in baseline platelet count by 30–
50% occurs generally within 5–14 days after primary exposure,
and can also be of rapid onset, developing within 24 hours in
patients who have had heparin exposure within the previous
3 months. Occasionally delayed-onset HIT can occur several
days to weeks after heparin exposure and caused by antibodies
that activate platelets independently of heparin. The platelet
count generally reaches nadir in the range 20,000–100,000/µL
and it is very unusual for it to drop below 10,000–15,000/µL
in contrast to some other drug-induced thrombocytopenias.
But this fall in platelet count usually recovers within 5–7 days
upon discontinuation of heparin.
Patients with HIT (> 50%) develop thrombosis, which can
occur in the arterial or venous systems, or both.7 Patients
may develop stroke, myocardial infarction, limb ischemia,
deep venous thrombosis, or ischemia of other organs. The
thrombotic complications may force limb amputation or
may prove fatal. Occasionally other manifestations like
skin lesions (erythema with or without necrosis) at sites of
heparin injections can occur. Since the rate of HIT-associated
thrombosis is so significant, it is of critical importance to
discontinue heparin therapy when the diagnosis of HIT is
suspected. Moreover, strong consideration should be given to
using an alternative (nonheparin) anticoagulant (e.g. a direct
thrombin inhibitor) to prevent thrombosis.7
 Chapter 31  Clinicopathological Syndrome in Immune-mediated Heparin-induced Thrombocytopenia  119
Figure 1  Pathophysiology of HIT
Diagnosis
The differential diagnosis of HIT is wide and includes sepsis,
multiorgan failure, the antiphospholipid syndrome and other
drugs that cause thrombocytopenia. Before the diagnosis
of HIT, a few of the differential diagnoses of unexpected
thrombocytopenia as summarized in Table 1 have to be
considered.
As such, HIT is a clinicopathological syndrome and the
diagnosis is based on a combination of clinical and laboratory
features. The following laboratory findings and clinical
features aid in the diagnosis of HIT.
Platelet Count Monitoring
Because the drop in platelet count is a primary way of
recognizing HIT, routine monitoring of the platelet count is
recommended for most patients receiving heparin treatment.
The American College of Chest Physicians, in collaboration
with the College of American Pathologists, have published
a guideline8 in relation to monitoring of platelet count in
patients at high risk for HIT. These recommendations are
summarized in Table 2. British guidelines3 recommend a
baseline platelet count before initiating heparin treatment in
all patients to allow estimation of relative changes. In higher
risk patients, such as individuals receiving unfractionated
heparin at therapeutic doses, the platelet count should be
checked at least every other day from day 4 to 14 of treatment
(or until heparin is stopped, whichever is sooner). In lower
risk surgical, medical and obstetric patients, monitoring
should be at least on every second to fourth day between
days 4 and 14 while on heparin treatment. Obstetric patients
receiving prophylactic doses of low molecular weight heparin
(LMWH) do not need routine platelet monitoring.
PF4 ELISA Assay
The laboratory tests are of two types, measurement of antibody
binding to PF4/heparin complexes or detection of heparin-
dependent activation of platelets by patient serum.4 The
120  Section 1  Clinical Hemotherapy

Table 1: Differential diagnoses of HIT

Diagnoses Differentiating features
Pseudothrombocytopenia Normal platelet count in citrated blood, platelet aggregates in peripheral blood smear
Nonimmunologic HIT After 1–2 days of therapeutic anticoagulation with UFH, platelet count rarely decreases <100,000/µL
(diagnosis by exclusion)
Massive pulmonary embolism Almost clinically indistinguishable from HIT, if occurring 5–14 days following start of heparin

DIC/Sepsis Often insidious onset, bleeding complications, consumption of clotting factors

Drug-induced thrombocytopenia Usually 7–10 days following introduction of a new drug. Platelets <20,000/µL, bleeding complications
Autoimmune thrombocytopenia Not associated with heparin medication
Diabetic ketoacidosis Acute thrombocytopenia with onset of illness

GP IIb/IIIa inhibitor-induced Begins within 12 hours of IIb/IIIa-inhibitor infusion, platelets <20,000/µL, bleeding complications
thrombocytopenia (important differential diagnosis: Pseudothrombocytopenia)

Post-transfusion purpura 7–14 days after transfusion in preimmunized patients (>95% women) , Platelets <20,000/ µL, bleeding
complications

Table 2: Consensus guideline for platelet count monitoring for HIT8

Population Example
Recent heparin exposure Patients starting UFH or LMWH and who received UFH within
the previous 100 days; patients whose heparin exposure
history is unknown
Acute, systemic reactions Patients with acute inflammatory, cardiorespiratory,
after intravenous UFH bolus neurological, or other unusual symptoms and signs within
30 min after an intravenous UFH bolus
Risk of HIT >1%† Patients receiving UFH at therapeutic doses
Postoperative patients receiving UFH antithrombotic
prophylaxis
Risk of HIT 0.1–1%† Medical/obstetric patients receiving prophylactic‐dose UFH,
or LMWH after first receiving UFH; postoperative patients
receiving prophylactic dose LMWH, or intravascular catheter
UFH flushes
Risk of HIT <0.1%† Medical/obstetric patients receiving only LMWH; medical
patients receiving catheter UFH flushes
*As recommended by College of American Pathologists. (Adapted with permission from Warkentin TE, Greinacher A. Heparin‐induced thrombocytopenia:
recognition, treatment, and prevention. Chest. 2004;126:311S–37S.8)
†Risk stratification is based on the overall incidence of HIT in different patient population
Monitoring guideline*
Obtain baseline platelet count and repeat
platelet count within 24 hours of starting
heparin
Obtain platelet count immediately to
compare with recent prior platelet counts
Monitor at least every 2 days until day
14 of treatment or until UFH is stopped,
whichever comes first
Monitor at least every 2 days between
postoperative days 4 and 14† or until UFH is
stopped, whichever comes first
Monitor every 2 or 3 days from days 4 to 14†
or until UFH is stopped, whichever comes
first, when practical
As clinically indicated (no routine
monitoring)

former is performed by ELISA and is highly sensitive but with a
specificity that ranges from 50 to 90%. In this target complexes
of PF4 and heparin or heparin-like molecules are immobilized
on a solid phase. To perform the test, the patient’s serum is
added to pre-made complexes of PF4 and heparin or heparin-
like molecules (e.g. polyvinyl sulfate, or PVS) alone and in
the presence of high-dose (100 U/mL) heparin. Heparin-
dependent antibody binds to the complexes and is detected
via enzyme-conjugated antihuman immunoglobulin. An
optical density value above 0.4 in the PF4-PVS well that can
be inhibited by high-dose heparin confirms the presence
of a heparin-dependent antibody in the patient’s sample.
Although IgG antibodies are the most clinically relevant
antibodies causing this syndrome,9 occasional patients with
HIT appear to have only non-IgG (IgM or IgA) antibodies.10
PF4 ELISA assays are available in two forms: those that detect
but do not differentiate IgG, IgM and IgA heparin-dpendent
antibodies, and those that detect only IgG.
 Chapter 31  Clinicopathological Syndrome in Immune-mediated Heparin-induced Thrombocytopenia  121
14C-serotonin Release Assay
In 14C-serotonin release assay (SRA), normal fresh target
platelets are incubated with 14C-serotonin, which is taken up
into the dense granules of platelets. The target platelets are
then exposed to the patient’s serum in the presence of low
and high concentrations of heparin. Release of at least 20% of
the radioactive label at the low dose of heparin and inhibition
of this release at the high dose confirms the presence of
heparin-dependent antibodies.
Other Functional Tests
Other functional tests used to detect heparin-dependent
antibodies include the heparin-induced platelet aggregation
test and the heparin-induced platelet activation test. PF4
ELISA and SRA are more sensitive and specific than the
platelet aggregation test in patients for whom there is clinical
suspicion. However, in asymptomatic patients receiving
heparin or in those who have not yet received the drug,
neither test is sufficiently predictive of HIT to warrant its use
in screening.7
Combining Clinical Assessment and
Appropriate Laboratory Testing
Combining clinical assessment and appropriate laboratory
testing is essential in the diagnosis of HIT.4 The first stage is
estimation of the pretest probability, for example, using the so-
called 4Ts scoring system which takes account of: (1) degree
of thrombocytopenia; (2) timing of thrombocytopenia; (3)
presence of thrombus or other clinical manifestations of HIT;
(4) Other potential causes of thrombocytopenia (Table 3).
In patients with a low pretest probability no laboratory
testing is required and heparin can be continued. If the EIA
is weakly positive at low heparin concentration and reactivity
is not inhibited at high concentration, HIT is unlikely and
heparin can be continued. (Abs interference in plt crossmatch
and other BTS service). A strongly positive IgG EIA indicates
Table 3: 4Ts scoring system of estimation of pre‐test probability of HIT
Category 2 points
Thrombocytopenia >50% fall, or nadir of 20–100×109/L
Timing of platelet count fall Days 5–10, or ≤1 day if heparin
exposure within past 30 days
Thrombosis or other Proven thrombosis, skin necrosis,
sequelae or, after heparin bolus, acute
systemic reaction
Other cause for None evident
thrombocytopenia
Points assigned in each of four categories are totalled, and the pre‐test probability of HIT by total points is as follows: 6 to 8 = high, 4 to 5 = intermediate; 0 to 3
= low. (Adapted with permission from Warkentin et al. Hematology/the education program of the American Society of Hematology. Copyright 2003, American
Society of Hematology.11)
an increased risk of platelet activating antibodies and a
platelet activation assay should be performed. If this is
positive, HIT is extremely likely. Finally, the clinical situation
should be reassessed to support or excluded the diagnosis.
Management
In HIT the heparin should be stopped immediately and
a non-heparin anticoagulant should be substituted.
Stopping heparin alone is insufficient as the risk of
thrombosis approaches 50% even in those who have isolated
thrombocytopenia and are clinically asymptomatic at the
time of diagnosis. Cross-reactivity between unfractionated
and low-molecular-weight heparin approaches 100% and
therefore the latter should not be used when HIT occurs in
patients receiving the former. Since initiation of vitamin K
antagonists may worsen the thrombosis associated with HIT,
these should be stopped and vitamin K administered. The main
non-heparin agents used are two direct thrombin inhibitors,
lepirudin and argatroban, and the heparinoid danaparoid.
Other agents that are sometimes used though not approved
in this setting are the direct thrombin inhibitor bivalirudin
and the synthetic pentasaccharide and factor Xa inhibitor
fondaparinux. Fondaparinux, a synthetic pentasaccharide
(smaller than LMWH) that is structurally related to the anti‐
thrombin binding site of heparin, is expected to be less likely
to induce HIT. The main disadvantage of these agents is that
they all carry a significant risk of bleeding and none has an
antidote. They each have advantages and disadvantages
in different clinical settings and expert advice should be
sought when considering their use. If oral anticoagulants
are required they should be initiated at low doses after the
platelet count has recovered to >1, 50,000/µL and overlapped
with one of the agents mentioned above for a minimum of
5 days and until the INR has been in the therapeutic range
for 48 hours. Platelet transfusions are contraindicated in HIT.
Plasmapheresis and use of intravenous immunoglobulin
has been helpful in anecdotal reports, but whether it is of
additional benefit to current treatments is unclear.
1 point 0 point
30–50% fall, or nadir of 10–19×109/L 30% fall or nadir <10×109/L
>Day 10 or unclear (but fits with HIT), or ≤1 day (no recent heparin)
≤1 day if heparin exposure within past
30–100 days
Progressive, recurrent, or silent None
thrombosis; erythematous skin lesions
Possible Definite
122  Section 1  Clinical Hemotherapy
Although exposure to heparin is usually avoided following
the diagnosis of HIT, the antibodies do not usually persist
beyond 100 days and re-exposure to heparin after this time
does not generally lead to recurrence of HIT.
Prevention
The incidence of HIT can be reduced by:
• Limiting courses of heparin to <5 days, if possible.
• Using LMWH in place of unfractionated heparin for
thromboprophylaxis in high‐risk postoperative patients.
• Porcine UFH is associated with lower incidence of HIT
than bovine UFH.
• In general, monitoring the platelet count at least every
other day between days 4 and 14 of heparin exposure or
until heparin is discontinued.
• Taking care not to automatically initiate heparin if a
patient is re‐admitted for a thrombotic event; the medical
record must be thoroughly reviewed for use of heparin on a
previous admission (within the past 100 days). Unfortu­
nately, in some cases, the previous heparin exposure
may not be recorded in the medical record (for example,
heparin flushes).
• Orders for use of heparin flushes and heparinized normal
saline must be written and signed by a physician and use
heparin flushes and heparinised normal saline must be
documented in the patient’s medical record
Key points
• Heparin-induced thrombocytopenia (HIT) is an immune‐
mediated event that can have severe life‐ and limb‐
threatening complications.
• Despite thrombocytopenia, bleeding is rare; rather, HIT is
strongly associated with thromboembolic complications.
• When a diagnosis of HIT is suspected immediate cessation
of all forms of heparin, including unfractionated heparin
(UFH), low molecular weight heparin (LMWH) and
heparin flushes, is imperative.
• Treatment of HIT should be initiated based on clinical
suspicion and must never be delayed pending laboratory
confirmation of HIT.
• A direct thrombin inhibitor, such as lepirudin, danaparoid
or argatroban, is considered the agent of choice for
treatment of HIT.
• Warfarin should not be used until the platelet count has
recovered.
Information to the Patient and
Record Keeping
The diagnosis of HIT should be clearly recorded in the
patient’s notes and marked as a serious allergy. The condition
should be clearly explained to the patient and an information
leaflet may be helpful in this respect. The patient should be
issued with an antibody card.
References
1. Franchini M. Heparin-induced thrombocytopenia: an update.
Thrombosis Journal. 2005;3:14.
2. Kelton JG, Smith JW, Warkentin TE, et al. Immunoglobulin
G from patients with heparin-induced thrombocytopenia
binds to a complex of heparin and platelet factor 4. Blood.
1994;83:3232–9, PubMed.
3. Keeling D, Davidson S, Watson H. Guideline: the management
of heparin‐induced thrombocytopenia. Br J Haematol.
2006;133:259-69, PubMed.
4. Greinacher A. Heparin-induced thrombocytopenia. J Thromb
Haemost. 2009;7(Suppl.1):9-12.
5. Kickler TS, Ness PM, Herman JH, Bell WR. Studies on
the Pathophysiology of posttransfusion purpura. Blood.
1986;68:347-50.
6. Christie DJ, Pulkrabek S, Putnam JL, et al. Posttransfusion
purpura due to an alloantibody reactive with glycoprotein 1a/
IIa (anti-HPA-5b). Blood. 1991;77:2785-9.
7. Seidenfeld AM, Owen J, Glynn MF. Post-transfusion purpura
cured by steroid therapy in a man. Can Med Assoc J.
1978;118:1285-6.
8. Warkentin TE, Greinacher A. Heparin-induced throm­
bocytopenia: recognition, treatment, and prevention:
the Seventh ACCP Conference on Antithrombotic and
Thrombolytic Therapy. Chest. 2004;126:311S–37S.
9. Nugent D, McMillan R, Nichol JL, Slichter SJ. Pathogenesis
of chronic immune thrombocytopenia: increased platelet
destruction and/or decreased platelet production. Br J
Haematol. 2009;146:585-96.
10. McMillan R. Therapy for adults with refractory chronic immune
thrombocytopenic purpura. Ann Intern Med. 1997;126:307-14.
11. Warkentin TE, Aird WC, Rand JH. Platelet–endothelial
interactions: sepsis, HIT, and antiphospholipid syndrome.
Hematology (Am Soc Hematol Educ Program). 2003.pp.497–
519.
Single Donor Platelets Versus
Random Donor Platelets

Rajni Bassi, Ajata Shatru Kapoor

32
Introduction One unit of platelet concentrate contains:
• Volume: 50–70 mL
Platelets are anuclear cytoplasmic fragments. The time • Platelets: ≥ 5.5 × 1010
interval from differentiation of the stem cell to the production • pH > 6.0
of platelets averages about 10 days. The production of • RBC contamination: Traces to 0.5 mL
platelets in a healthy person is approximately 40 × 109/L/day. • WBC contamination: 5.5 × 107– 5.5 × 108.
The normal platelet count is about 150–400 × 109/L. Platelets
help in formation of primary hemostatic plug and to maintain
Buffy Coat Removal Method of Preparation of
normal hemostasis. The hemostatic plug provides the surface
on which fibrin plug forms which results in cessation of Platelet Concentrate
bleeding, correction of prolonged bleeding time and rise in Platelets are prepared by using top and bottom bags. Whole
platelet count. blood can be stored at room temperature for up to 24 hours
prior to platelet harvesting. Hard spin is first done to spin
platelets into buffy coat on a cushion of red cells and then
Preparation of Platelet using a slow spin to isolate the platelets from the separated
concentrate1 buffy coat.3
• One unit of platelet concentrate is prepared from single
Random Donor Platelet (Whole Blood buffy coat.
Derived Platelets) • Pooled platelet concentrate are prepared from pooling of
4–5 buffy coats.
• Platelet rich plasma–platelet concentrate (PRP–PC).
• Buffy coat removal method of platelet concentrate (BC–
PC). Advantages of BC–PC over PRP–PC
BC–PC PRP–PC
Single Donor Platelet (SDP) Minimum red cell contamination. Red cell contamination is more
• Prepared by apheresis or cell separator. Harvesting of platelets from a Harvesting of platelets on
biological cell bed, hence better plastic surface, hence lesser
in vivo survival in vivo survival
Random Donor Platelets Lower leukocyte contamination More leucocyte contamination
Improvement and Less standardization of quality
Platelet Rich Plasma–Platelet Concentrate2 standardization of quality of of products
Platelet rich plasma–platelet concentrate (PRP–PC) are products
prepared from whole blood kept at room temperature (20–24°C)
and within 6–8 hours of collection. 350–450 ml of whole blood
is collected and stored at room temperature. Within 6 hours,
Advantages of Pooled Buffy Coat Method
blood is centrifuged with a light spin and PRP is separated into • BC–PC pooling provides a high dose platelet concentrate.
satellite bag. PRP is centrifuged again with a heavy spin and equivalent to apheresis dose platelets (pooled 5 BC–PC
separated into PC and fresh frozen plasma (FFP). have 3 × 1011 platelets).
124  Section 1  Clinical Hemotherapy
• The BC–PC are leukodepleted and contains 5 × 106 leuko-
cytes in 200–300 ml of plasma.
• Cost-effective when compared with plateletpheresis and
can be considered as an alternative when eligible donors
for SDP are not available.
One unit of BC–PC contains:
• Volume: 70–90 mL
• Platelet count: 6–9 × 1010
• pH > 6
• WBC contamination: >5.5 × 106
• RBC contamination: Traces to 0.5 ml.
Dose of Random Donor Platelet
• One unit per 10 kg body weight or 4–6 units in an adult of
70 kg.
• One unit of RDP will raise the platelet count 5–10 × 109/L in
an adult of 70 kg body weight.4
Single Donor Platelets/
Plateletpheresis
Apheresis (or hemapheresis) is a Greek word that means to
separate or remove. In apheresis blood is withdrawn from a
donor or patient in anticoagulant solution and separated into
components. One (or more) component is retained and the
remaining constituents are returned to the individual. Any
one of the components of blood can be removed and the
procedures are specified for the component selected. The
process of retaining platelets is termed plateletpheresis, red
cells as erythrocytapheresis or leukocytes as leukapheresis,
etc. Apheresis can be nontherapeutic or therapeutic.
In most apheresis equipments, centrifugal force separates
blood into its components based on differences in specific
gravity. Blood is drawn from an individual with the assistance
of a pump. Anticoagulant (ACD) is added to the tubing to
prevent clotting. Anticoagulated blood from a donor or
patient is pumped into a rotating bowl, chamber, or tubular
rotor in which layering of the components occurs based on
density. The desired component is retained and the remaining
elements are returned to the donor or patient by intermittent
or continuous flow.5
General Requirements for Apheresis4
• A qualified, licensed physician is responsible for all aspects
of apheresis program
• Equipment should be in good working condition
• Well trained and motivated staff
• Operator must know all aspects of its operation and
trouble shootings
• An apheresis operator must be able to relieve the anxiety
of donor/patient
• Records should be maintained.
Specific Criteria for Selecting
Plateletpheresis Donor4
• Donor undergoing an occasional apheresis procedure
must meet the same criteria as whole blood donation.
Donor should be preferably repeat donor. Written consent
of the donor is taken after explaining the procedure and its
adverse effect.
• Venous access is an important consideration in apheresis
donor and vein should be examined at the time of selection
of a donor as:long needle in and out time, prolonged
flow rate and frequent need for two venepuncture with
continuous flow equipment.
• Donor should be screened prior to apheresis for markers
of infectious diseases transmitted by blood and its
component. If donor is undergoing repeat procedure
within 30 days interval, testing of infectious markers of
disease may not be needed.
• Routine hemogram, ABO group, Rh type and screening for
unexpected antibodies are done. Platelet count must be
more than 150 × 109/L.
• Donors should not have taken aspirin containing
medication 36 hours prior to procedure.
• Platelets may be collected from HLA matched donors.
• The interval between the procedure should be at least
48 hours. A donor shall not undergo the procedure more
than 2 times in a week or 24 times in a year.
Donors Who Participate in Serial
Apheresis Program4
• Interval between two procedures should be at least 48 hours
and loss of red cells should not exceed 25 ml per week.
• If donors red cell could not be reinfused, or if the
participant donates a unit of whole blood, 12 weeks should
lapse before subsequent apheresis procedure.
• Careful monitoring of weight, blood cell count, serum
protein level and quantitation of immunoglobulin is
required. Serum protein should be > 6 g/dl.
Procedure
In plateletpheresis procedure, a portion of the donors platelet
and some amount of plasma is removed with the return of the
donors RBCs, WBCs, and remaining plasma. Apheresis machine
allows repeated draw-centrifuge-return cycle to obtain more
platelets. A routine plateletpheresis procedure usually takes
1–1.5 hours. The product is prepared in a closed systems and
can be stored for 5 days. The number of platelets in apheresis
product is equivalent to 6–8 random donor platelet concentrate.
Adverse Effects of Apheresis in Donor
• Citrate effect: Numbness and tingling sensation around
mouth.
• Citrate toxicity: Muscle cramps, shivering, nausea, vomit­
ing and tetany.
 Chapter 32  Single Donor Platelets Versus Random Donor Platelets  125

Table 1: Relative merits of single donor platelets and random donor platlets
Single donor platelets Random donor platelets
Platelets > 3 × 1011 (equal to platelets obtained from 5–6 whole blood 5.5 × 1010 platelets
donations)
Plasma volume 200 ml Plasma volume 30–40 ml
Leukocytes <5.5 × 106 Leukocytes 5.5 × 108 in each unit
Obviates the need of filtration Filtration is required to reduce leukocytes
Red cells—traces Red cells—more
pH-6.0 or more pH-6.0 or more
Exposes a patient to one donor Exposes a patient to multiple donors
Less exposure to infection More exposure to infection
Low-risk to alloimmunization Relatively more risk to alloimmunization
HLA- or platelet-matched donor product can be prepared for the Not possible
patients who have become refractory to platelets
Decreased risk of bacterial contamination and easy handling More risk of bacterial contamination platelets are pooled
Sophisticated equipment required Routine donation, can be made from whole blood
Highly trained persons required Not much

The problem is usually solved by decreasing the rate • Purer product

of infusion of returned component or giving the donor • Ability to match donor to patients—platelet matched or
exogenous calcium. HLA matched
• Fewer donor reactions due to-return of fluid-crystalloid
One unit of plateletpheresis contains: and anticoagulant
• Volume: 200–300 ml – Longer period in apheresis help refilling of intravascular
• Platelet count: ≥ 3.0–7.0 × 1011 components, with fluid from interstitial spaces
• pH > 6.0 • Smaller needle technology—venous access injuries are less.
• Residual leukocyte: < 5.0 × 106
• Red cell: Traces to 0.5 ml.
References
1. Moroff G, Holmes S. Concepts about current conditions for
Dose of Single Donor Platelets the preparation and storage of platelets. Transfus Med Rev.
One unit of SDP in an adult of 70 kg. One unit of platelet from 1991;5:48-59.
an apheresis donor will raise the platelet count of an adult of 2. Fisk JM, Pisciotto PT, Synder EL, et al. Platelet and related
products. In: Blood Banking and Transfusion Medicine.
70 kg body weight approximately by 30–60 × 109/L.4
Edition. Hillyer CD, Silberstein LE, Ness PM, Anderson KC,
Roback JD (Eds). Churchill Livingstone, Philadelphia. 2007.
Advantages of Single Donor Platelets5 pp.308-41.
3. Fijnheer R, Veldman HA, van den Eertwegh AJ, et al. In vitro
(Table 1) evaluation of buffy-coat-derived platelet concentrates stored
in a synthetic medium. Vox Sang. 1991;60:16-22.
• Reduced multiple donor exposure
4. Blood component preparation and their uses. In: Transfusion
– Reduced risk of alloimmunization Medicine Technical Manual. 2nd edn. Saran RK (Ed). 2003.
– Reduced incidence of transfusion transmitted infections pp.193-28.
• Full and effective transfusion dose 5. Apheresis. In: Transfusion Medicine Technical Manual 2nd edn.
• Higher quality product—leukoreduced products Saran RK (Ed). 2003;229-43.
Prophylactic Platelet Transfusion
Poonam Singal
Patients with severe thrombocytopenia are presumed to
be at increased risk for bleeding, and consequently, it has
been a standard practice for the past four decades to give
allogenic platelet transfusions to severely thrombocytopenic
patients as supportive care.1 Thrombocytopenia can result
from myriad causes including marrow suppression e.g. due
to drugs, irradiation, infiltration, intrinsic marrow disease
such as myelofibrosis, massive transfusion (dilutional
thrombocytopenia), or immune destruction (e.g. immune
thrombocytopenic purpura, thrombotic thrombocytopenic
purpura, neonatal alloimmune thrombocytopenia). Patients
with congenital or acquired platelet disorders, such as
Bernard-Soulier syndrome or platelet storage pool deficiency,
may also benefit from platelet transfusion. These individuals
will frequently have normal platelet counts but diminished
platelet function that is inadequate for normal hemostasis.2
Platelet transfusions may be given either prophylactically
to reduce the risk of bleeding, in the absence of clinical
hemorrhage (prophylactic transfusions) or to control
active bleeding when present (therapeutic transfusions).
Platelet transfusions are not indicated in all causes of
thrombocytopenia and may indeed be contraindicated in
certain conditions. Thus, the cause of the thrombocytopenia
should be established before a decision about the use of
platelet transfusion is made. Any decision must also be based
on an assessment of risk versus benefit. Risks associated with
platelet transfusions include alloimmunization, transmission
of infection, allergic reactions and transfusion-related acute
lung injury; potential benefits include reducing morbidity
associated with minor hemorrhage and reducing morbidity/
mortality resulting from major bleeding.3 While there is a
universal consensus to give platelet transfusions in the face
of severe bleeding, controversy persists as to the optimal dose
of platelets to use, and the appropriate platelet count level at
which to transfuse the nonbleeding patient. It is now generally
accepted that the decision to transfuse platelets or any blood
product should almost never be based solely on transfusion
thresholds (or transfusion triggers). Patient factors should
always be considered when deciding when an individual
patient should be transfused. These factors may include the
33
patient’s primary disease, the presence of fever or sepsis,
concurrent medications, and overall coagulation status. The
transfusion trigger varies in different clinical settings as given
here.
Transfusion Triggers
Prophylaxis for Surgery3
• Bone marrow aspiration and biopsy may be performed in
patients with severe thrombocytopenia without platelet
support, provided that adequate surface pressure is
applied.
• For lumbar puncture, epidural anesthesia, gastroscopy
and biopsy, insertion of indwelling lines, transbronchial
biopsy, liver biopsy, laparotomy or similar procedures, the
platelet count should be raised to at least 50 × 109/L.
• For operations in critical sites such as the brain or eyes, the
platelet count should be raised to 100 × 109/L.
It should not be assumed that the platelet count will
rise just because platelet transfusions are given, and a
preoperative platelet count should be checked to ensure that
the above thresholds have been reached.
Chronic Stable Thrombocytopenia
In patients who are chronically thrombocytopenic due to
chemotherapy, bone marrow transplantation or for marrow
conditions resulting in thrombocytopenia such as aplasia or
myelodysplasia:
• A threshold of 10 × 109/L is as safe as higher levels for
patients without additional risk factors. Risk factors
include sepsis, concurrent use of antibiotics or other
abnormalities of hemostasis.
• For patients without any risk factors, a threshold of
5 × 109/L may be appropriate, if there are concerns that
alloimmunization could lead to platelet refractoriness.
However, accurate counting of low platelet numbers may
create difficulties when trying to reduce the threshold
below 10 × 109/L.

A specific threshold for transfusion may not be appropriate
for patients with chronic stable thrombocytopenia who
are best managed on an individual basis depending on the
degree of hemorrhage.
Massive Transfusion and Disseminated
Intravascular Coagulation
A platelet count of around 50 × 109/L should be maintained.
In chronic disseminated intravascular coagulation (DIC),
or in the absence of bleeding, platelet transfusions should not
be given merely to correct a low platelet count.
Cardiopulmonary Bypass Surgery
Cardiopulmonary bypass (CPB) results in the destruction
of a significant fraction of the patient’s platelets. In addition
there is evidence that the remaining platelets may have their
function impaired by the bypass procedure. Further:
• There is no place for prophylactic transfusion of platelets
in patients undergoing CPB but platelets should be readily
available in all institutions undertaking cardiac surgery.
• The preoperative assessment of patients attending for
cardiac surgery should include a thorough review of all
medications likely to interfere with platelet function.
• The decision to transfuse platelets in patients following
CPB remains a clinical decision based on the evidence
of microvascular bleeding in association with excessive
postsurgical blood loss.
Bone Marrow Failure (Due to Disease,
Cytotoxic Therapy or Irradiation)
Prophylactic platelet transfusions have become standard
practice for patients with bone marrow failure. The platelet
count cut off for prophylactic platelet transfusion is deter­
mined in part by the concept that while thrombocytopenic
patients may develop petechiae and ecchymoses (dry
bleeding) they will not develop fatal hemorrhagic
complications without first developing extensive mucous
membrane bleeding (wet bleeding). It is recommended to:
• Transfuse platelets at count <10 × 109/L in patients with no
or only “dry” bleeding.
• Prophylactic threshold should be 20 × 109/L in patients
with risk factors like infection, fever or prior to minor
surgery.
Platelet Function Disorders
Patients with platelet function disorders rarely need platelet
transfusions. Even patients with severe inherited platelet
function disorders such as Glanzmann’s thrombasthenia
only have sporadic bleeding and may have no bleeding
for many years. Negligible or no excessive bleeding can be
expected in patients with acquired platelet function disorders
as the impairment in platelet function is much less than in
Chapter 33  Prophylactic Platelet Transfusion  127
Glanzmann’s thrombasthenia. However, acquired causes of
platelet dysfunction can exacerbate bleeding in patients who
already have impaired hemostasis.
It is recommended that for prophylaxis before invasive
procedures for patients with a known or suspected platelet
function disorder:
• Withdraw drugs known to have antiplatelet activity
• Correct any underlying condition known to be associated
with platelet dysfunction, if possible
• Correct the hematocrit to > 0·30 I/L in patients with renal
failure
• Consider the use of Desmopressin DDAVP, in patients
with inherited dysfunction defects
• Consider the use of DDAVP or cryoprecipitate in patients
with uremia
• Use platelet transfusions where the above methods are not
appropriate or are ineffective.
Another important aspect of prophylactic platelet
transfusion therapy is regarding the dose of platelets to
be transfused. In practice, a single dose of transfused
platelets generally contains approximately 3 × 1011 platelets
corresponding to 1 apheresis derived (AD)-platelet product
or 5 to 6 pooled whole blood (WB)-platelet units. The optimal
adult dose of platelets has not been clearly established, and
doses are often determined based on factors unrelated to
efficacy such as cost and availability. For example, an adult’s
body weight is not usually considered when determining
the number of platelets to administer. In general, the higher
a patient’s post transfusion platelet increment, the longer
the interval will be before the next platelet transfusion
is required. Endogenously produced platelets normally
survive approximately 9–10 days in the absence of diseases
that decreases platelet survival. Transfused platelets do not
circulate this long in thrombocytopenia patients, as they are
more rapidly consumed, for instance, to maintain vascular
integrity. At platelet count below 100 × 109/ L, a direct
relationship exists between low platelet count and shorter
platelet life span in the circulation.
Large/Infrequent Versus Small/Frequent
Platelet Dosing Strategies2
Several transfusion specialists advocate transfusing doses of
platelets larger than the standard 6 WB platelet pool albeit
at longer intervals between transfusions, e.g. 10–12 WB-
platelet units every 2–3 days. The rationale for this practice
is largely based on decreasing the number of individual
transfusion episodes. This is more convenient in outpatient
settings. As expected, it is possible to obtain a higher post
transfusion platelet increment by transfusing higher platelet
numbers. However, it is unclear whether this practice is
more effective than standard platelet doses in reducing the
risk of spontaneous bleeding. Alternatively smaller doses of
platelets transfused at shorter intervals (e.g. 3–4 WB-platelets
per transfusion) may reduce the total number of platelets
required during a patient’s thrombocytopenic period. This
128  Section 1  Clinical Hemotherapy
strategy could decrease the overall number of platelets
needed for a large patient population and decrease the
number of exposures for an individual patient. Thus, smaller
platelet doses may be more economical but this practice
has been criticized for possibly increasing the number of
individual transfusions, which increases overall costs.4
Another factor which favors the lowering of transfusion
trigger and low platelet dose is the role of thrombopoietin.
Thrombopoietin has been recognized as the major hema­
topoietic growth factor that stimulates platelet production
and it is constitutively made in the liver at a constant rate.
TPO binds to its ligand on the surface of platelets and
megakaryocytes and in the absence of megakaryocytes or
platelets, TPO levels rise. It has been documented that TPO
is adsorbed on the surface of transfused platelets. Therefore,
reductions in the number of transfused platelets by decreasing
the platelet transfusion trigger and the platelet dose should
further reduce the number of platelet transfusions required as
elevated TPO level will stimulate platelet production as soon
as megakaryocytes appears in the marrow. With endogenous
TPO stimulated megakaryocyte platelet production the
duration of patients thrombocytopenia will be reduced,
further decreasing the need for transfused platelets.5
Therefore, an optimal dose of platelets has not been
clearly defined, and transfusion practices will likely remain
largely based on local preferences which will often be heavily
influenced by costs and supply.
References
1. Blajchman MA, Slitcher SJ, Heddle NM, et al. New strategies
for optimal use of platelet transfusions. Haematology Am Soc
Haematol Edu Program. 2008.pp.198-204.
2. Fisk JM, Pisciotto PT, Snyder EL, et al. Platelets and Related
Products. Blood Banking and Transfusion Medicine, 2nd edn.
Churchill Livingstone. 2007.p.326.
3. Guidelines for the use of platelet transfusions. British Journal
of Haematology. 2003.pp.10-23.
4. Ackerman SJ, Klumpp TR, Guzman GI, et al. Economic
consequences of alterations in platelet transfusion dose:
analysis of a prospective randomized, double blind trial.
Transfusion. 2000;40:1457-62.
5. Slichter SJ. Evidence based platelet transfusion guidelines.
Haematology Am Soc Hematol Educ Program. 2007.pp.172-8.
 Apheresis
Maximizing the Resources:
Multicomponent Collection
Prashant Agarwal
There are several challenges in the field of transfusion medi­
cine in several countries. These are, shortage of blood supply
which may be due to less motivation among the population
about blood donation, more need of specific components
as compared to blood donation, various regulatory issues,
public pressure from safety of transfusion transmitted
infections and cost of blood components. So, in the current
scenario of transfusion medicine it is very necessary to
advance and implement the technology which can collect
multiple components.1
Now-a-days the main aim of transfusion medicine are self-
sufficiency, good quality of blood components, blood donor
health and satisfaction of patient at the lower cost. Aging of
the population, more stringent donor criteria, economic
and social challenges and increasing complexity of modern
medicine all are responsible for the blood shortage. These
are hardly compensated by blood collection unless multi-
component collection (MCC) strategies are simultaneously
developed. MCC is an exciting concept in modern era of
transfusion medicine but switching donors from conventional
blood collection to individual components collection which
is termed as apheresis technology, is not so easy.2
There are several studies which states that over 95% of
donors can be switched to MCC and only 15 to 20% refuse
to enlist in MCC programme. But in reality only from 15 to
20% of eligible donors undergo MCC for reasons ranging from
structure limitations, poor organization of blood drives, lack
of information, limited number of machines, lack of qualified
personnel and poor appreciation of cost involved. Another
reason why MCC is underutilized is because it took years for
the industries involved in apheresis to fully understand the
advantage of MCC in terms of potential for expansion of their
market.2
Multi-component apheresis (MCA)—Apheresis techno­logy
is well established in modern era of transfusion medicine. It
was first incepted in 1970s. It is widely used to collect platelets,
plasma and red cells from allogeneic donors although
autologous donation are also utilized. With the technology
34
improvement, it is possible to collect multiple doses of
blood element (e.g. platelets, RBCs) or combination of blood
components (RBCs and plasma; platelets and RBCs).1
Donation criteria—The requirement of donors for MCA are
same as whole blood donation except there are some specific
requirements. These are: (1) Extracorporeal volume should not
exceed 13% of total blood volume, (2) If we plan to collect one
or more plateletpheresis units, previous platelet count should
not be less than 150 × 10 9/L, (3) To obtain more than one
plasma unit, donor should have a total amount of proteins over
6 g/dL, (4) Erythropheresis require Hb more than 12.5 g/dL for
women and 13.5 g/dL for men in order to obtain a single unit.
For double erythropheresis requirements are: volume over
5000 mL and hemoglobin over 14 g/dL and hematocrit >42%.
Donor selection: Due to shortage of donors it is possible to
select a special pool of donors for different routine protocols,
e.g.
• B and AB donors are better for plasma and platelets.
• O and A negative are interesting for RCC (single or double
dose)
• Donors with blood volume > 5000 mL and platelet count
> 250 × 109/L can be reserved for double platelet protocols.
• Donors with blood volume > 4500 mL and platelet count
> 270 × 109/L can be used for double dose of platelets, or
single dose of platelets and a red cell concentrates.
Blood components: The yield of units should be set to provide
maximum benefit to the patients without compromising
donor’s health, e.g.
• Unit of platelets: 3.5 × 1011 per unit
• Double platelets: 7 × 1011 per unit
• Unit of plasma: 230 mL
• Double plasma: 460 mL
• RCC: 180 mL.
Now-a-days it is possible to collect apheresis units of
platelets and red cells leucodepleted, HLA and HPA matched
donors for patients with alloantibodies and more volume of
plasma for donors like IgA deficiency.3
130  Section 1  Clinical Hemotherapy
Multi-component connection has advantages like more
frequency of donation, more donor safety (less vasovagal re­
action), enhance regula­tory compliance because by collecting
double units of plate­lets or red cells, less inventory management
is needed, less donor exposure by double units and collection
of product from special donors. MCA is one of the tools that
enables blood collectors to meet the challenges of supply and
better quality.1
References
1. Mark A. Popovsky: Multicomponent apheresis blood collection
in the united states; current status and future direction.
Transfusion and Apheresis Science. 2005;32:299-304.
2. M Valbonesi, et al. Multicomponent collection as of 2005.
Transfusion and Apheresis Science. 2005. pp. 287-97.
3. Lydia Blanco. Tailored collection of multicomponent by
apheresis. Transfusion and Apheresis Science. 2002. pp. 123-7.
Therapeutic Plateletpheresis
Tulika Chandra
Introduction
Plateletpheresis (more accurately called thrombocytapher­
esis or thrombapheresis, though these names are rarely
used) is the process of collecting thrombocytes, more
commonly called platelets, a component of blood involved in
blood clotting. The term specifically refers to the method of
collecting the platelets, which is performed by a device used in
blood donation that separates the platelets and returns other
portions of the blood to the donor. Platelet transfusion can
be a life-saving procedure in preventing or treating serious
complications from bleeding and hemorrhage in patients
who have disorders manifesting as thrombocytopenia (low
platelet count) or platelet dysfunction. This process may
also be used therapeutically to treat disorders resulting in
extra ordinarily high platelet counts such as essential throm­
bocytosis.
Therapeutic plateletpheresis
This is occasionally used in patients with myeloproliferative
disorders and symptomatic thrombocytosis until chemo­
therapy and/or antiplatelet drug therapy takes effect.
Thrombocytosis and the Rationale for
Therapeutic Plateletpheresis (TP)
Thrombocytosis may occur in 3 settings:
1. Reactive thrombocytosis in response to splenectomy, Fe
deficiency, acute hemorrhage, chronic inflammation, and
malignancy.
2. In myeloproliferative disorders such as CML or essential
thrombycythemia.
3. Rare inherited forms.
Symptoms are only seen in patients with myeloproliferative
disorders, including both thrombosis and hemorrhage.
• Hemorrhage: Due to abnormal platelet function
• Thrombosis maybe arterial or venous.
35
Cerebrovascular events most common clinical finding.
Complications not directly related to platelet count, can be
seen with platelet counts between 500 × 109 – 5000 × 109/L.
Many patients are asymptomatic despite high counts.
Thromboses more common in the elderly (> 60) or those with
cardiovascular risk factors, including previous episodes.
Therapeutic plateletpheresis achieves rapid reduction
of platelet count, and is usual­ ly reserved for patients
with acute serious thrombotic or hem­orrhagic events, or
high risk patients with very high platelet counts (> 1000
× 109/L) (ASFA/AABB Category I Indication). Follow-
up procedures can help maintain the lower count
until pharmacologic agents start to take effect. Pharmaco­
therapy (hydroxyurea, anagrelide) eventually required to
lower platelet count.
Practical Considerations of TP
• Can be performed with any centrifugal apheresis
instrument.
• In emergent cases, peripheral venous access can be used
instead of central venous catheter (and avoid the attendant
risks of catheter placement).
• Instrument configuration, anticoagulation, etc. same as
in a platelet donation, however, a higher flow rate in the
removal line may be desirable.
• Large volume of cells must be removed. Available data
suggest that 30–50% of circulating platelets can be
removed after processing ~ 1.2 blood volumes over a wide
range of platelet counts. STAT intraprocedural count may
be helpful.
• No consensus on the target platelet count after procedure.
Sample Therapeutic Plateletpheresis Orders
When ordering plateletphersis, following orders should be
considered:
• Procedure target:
– Volume: 2 × TBV (total blood volume).
132  Section 1  Clinical Hemotherapy
• Frequency of procedure:
– Daily until platelet count < 100 × 109/L.
• Anticoagulation:
– ACD-A
• Medication orders:
– Ca gluconate to prevent citrate reaction symptoms.
– Ca gluconate 10%: Normal saline IV piggyback PRN
citrate reaction.
– Ca gluconate 10%: Normal saline during treatment.
• Central venous access care:
– Flush each lumen of catheter with 10 mL NS followed
by Heparin + NS mL to equal internal volume of each
lumen.
– Flush each lumen of catheter with 10 mL of NS followed
by ACD-A to equal the internal volume of each lumen.
• laboratory orders:
– Preprocedure: CBC with platelet count.
– Postprocedure: CBC with platelet count.
• Standing orders:
– Two CaCO3 tablets (e.g. Tums) PO/PRN every 30 minutes
for citrate symptoms.
– Notify consultant doctor for BP < 90/50 mm Hg or
> 180/100 mm Hg and for pulse rate < 50 minutes or
> 150 minutes
– NS IV: 250 mL × 2 and/or 250 mL 5% albumin for
hypotension (BP systolic < 90 mm Hg) PRN.
– Use blood warmer.
– Normal saline: 1–2 L to prime, rinse-back and infuse
during treatment.
Therapeutic plateletpheresis is generally reserved for
patients with myeloproliferative disorders and hemorrhage or
thrombosis associated with an increase in circulating platelets.
Many centers consider using plateletpheresis when the
patient’s peripheral platelet count is greater than 1000 × 109/L,
although no consistent relation between the level of platelet
elevation and the occurrence of symptoms has been found, and
no generally accepted assay of platelet dysfunction predicts
which patients are at risk. A single cytapheresis procedure can
lower the platelet count by 30 to 50%. Plateletpheresis can have
dramatic effects for selected patients such as those with evolving
digital gangrene. Attempts to maintain thrombocythemic
patients at normal platelet counts by cytapheresis alone have
not been successful; more practical long-term chemotherapy
should be instituted concurrently.
suggested reading
1. Joint United Kingdom (UK) Blood Transfusion and Tissue
Transplantation Services Professional Advisory Committee.
2014.
2. Therapeutic plateletpheresis in thombocythemia. Transfusion.
1979;19(2):147-52.
Therapeutic Plasma Exchange
Aseem K Tiwari
Apheresis is the process in which whole blood is withdrawn
from a person’s circulation, a component such as plasma is
separated out and retained, and the remainder is returned to
the patient usually with some replacement fluid.
Apheresis may be performed in a healthy blood donor to
harvest a blood component like platelets, red cells, plasma or
even hematopoietic stem cells or apheresis may be performed
in a patient as a therapy and is called therapeutic apheresis.
Therapeutic apheresis is a branch of transfusion medicine
encompassing the treatment of diseases through removal of
blood components or specific blood substances.
The goal of therapeutic apheresis is to remove a pathologic
element from the blood. The element may be a plasma
protein such as an autoantibody as in myasthenia gravis, red
cells as in sickle cell crisis, leukocytes as in hyperleukocytosis
accompanying acute leukemia, or platelets as in marked
thrombocytosis. Therapeutic apheresis is sometimes classified
as therapeutic cytapheresis and therapeutic plasmapheresis.
Therapeutic cytapheresis is the removal of cellular blood
elements. If red cells are removed and replaced with donor
red blood cells (RBCs), it is called red cell exchange. Thera­
peutic plasmaphereis is the separation and removal of plasma
from whole blood with replacement by a colloid solution
(typically albumin or plasma) or a combination of crystalloid/
colloid solution. It is also called therapeutic plasma exchange
(TPE). So the term therapeutic plasmapheresis and thera­
peutic plasma exchange (TPE) is used synonymously. TPE is
also the most common therapeutic apheresis modality. The
effectiveness of TPE depends on concentration of element in
blood, volume of blood processed and equilibrium between
blood and the extra-vascular volume of distribution. The
principles of TPE are as follows:
• Apheresis is most efficient at the beginning of the
procedure and least efficient at the end.
• Exchange of a single blood volume will eliminate
approximately two-thirds of a substance (if it does not
move from extravascular sites to the intravascular space).
36
• Continued production of the substance or mobilization
from tissues into the intravascular space will result in less
apparent reduction despite an equal or even greater total
amount removed.
For a substance that is strictly intravascular, 36.8% of the
initial concentration will remain after a single blood volume
exchange by TPE. However, if the substance is also present
in the extravascular space with a total volume of distribution
equal to twice the blood volume, for example, 60.6% will
remain after processing a single blood volume. Generally
1–1.5 volume exchange is performed every alternate day
for most of the disease conditions. However, the exchange
volume and frequency may both change depending on the
disease to be treated.
It is very crucial to understand as to which are the right
indications for TPE. American Society for Apheresis (ASFA)
has done a very good classification of indications for TPE.
Although there are many case reports of successful treatment
of a large variety of disease and conditions by apheresis,
there have been few high-quality trials for justifying TPE in
various indications. ASFA has published a categorization of
indications for apheresis based on the best available evidence
Table 1.
We would like to share our experience in TPE in last few
years. Ideally, physicians ordering therapeutic apheresis
procedures on their patients for various indications should
be aware of the “right” indications. However, actually many
practicing physicians commonly overuse, under-use, and
misuse this therapeutic intervention. Transfusion medicine
specialists who perform this therapy on patients may steer
the treating physicians in right direction through continuing
medical education (CME) which gives them an opportunity
to keep abreast with the latest “right” criteria of ordering
therapeutic apheresis.
We at department of transfusion medicine in Gurgaon
reviewed, collected, and interpreted the effect of formal CME
interventions (conferences, clinical rounds, meetings and
134  Section 1  Clinical Hemotherapy
Table 1: Categorization of indication of therapeutic plasma
exchange
Category Description
I Disorders for which apheresis is accepted as first-line
therapy, either as a primary standalone treatment or
in conjunction with other modes of treatment
II Disorders for which apheresis is accepted as second-
line therapy, either as a standalone treatment or in
conjunction with other modes of treatment
III Optimum role of apheresis therapy is not established.
Decision making should be individualized
IV Disorders in which published evidence demonstrates
or suggests apheresis to be ineffective or harmful.
IRB approval is desirable if apheresis treatment is
undertaken in these circumstances
one-to-one interaction) on “ordering” practice of physicians
in context of therapeutic apheresis procedures. Different
CME interventions that incorporated didactic sessions
and practical experiences to teach about the indications,
clinical and managerial aspects of therapeutic apheresis
were conducted in the first quarter of 2012. Sessions mainly
involved teaching the practicing physicians about guidelines
and recommendations for use of therapeutic apheresis
as per American Society for Apheresis guidelines of 2010.
Data was collected and changes in ordering practice related
to therapeutic apheresis procedures before and after the
intervention were analyzed.
Total of 73 physicians from different departments were
involved in these interventions. Total of 589 therapeutic
apheresis procedures [therapeutic plasma exchange (TPE),
double filtration plasmapheresis (DFPP), leukocytapheresis
(LCP), RBC exchange] were performed during the entire
period between year 2010 to 2014. 214 procedures were
performed before intervention for 49 different patients while
375 procedures were performed for 84 different patients
after intervention. There was a significant improvement in
“ordering” practice of physicians in context of therapeutic
apheresis procedures; before intervention 38% of the patients
were from category I indications, 22% were from category II,
12% were from category III and 3% were from category IV,
while 20% of patients did not belong to any category. After
intervention there was significant improvement in indications
of category I 64% with a p value of <0.0001, category II (16%),
category III (7.1%) and category IV (1%). There was also
reduction in procedures not belonging to any category (10%).
Change in practice among the physicians was also observed in
the context of duration of therapy, discontinuation of therapy,
number of procedures and replacement fluid. It was concluded
that CME intervention appears to be effective at acquisition
and retention of knowledge, attitudes, skills, behavior and
clinical outcomes. Our data shows evidence that interactive
CME sessions enhance participant activity and provide the
opportunity to practice skills can effect change in professional
practice and therefore possibly patients outcomes.
Plasma Exchange as an Alternative
to Immunoglobulin
A range of indications currently treated with IVIg can use
therapeutic plasma exchange (‘TPE’) as an alternative.
A comparison of IVIg and TPE demonstrates that in
many cases TPE can be a clinically appropriate first-line
treatment. TPE is less expensive than IVIg in most of the
largest-consuming indications where TPE is an alternative.
Increasing availability of TPE could release several months
supply of IVIg for critical patients each year.
The clinical guidelines suggest TPE as an alternative to IVIg
for the following key indications:
• CIDP
• Guillain-Barré syndrome
• Myasthenia gravis
• Dermatomyositis
• Paraproteinemic polyneuropathies
• Polymyositis
• Systemic vasculitides and ANCA disorders.
Therapeutic plasma exchange reduces the effect of harmful
antibodies by isolating and replacing part of the patient’s
blood plasma. When blood is removed from the patient;
plasma is isolated and replaced with a substitution fluid such
as albumin it decreases the level of antibodies in the blood,
suppressing immunity. Effects can be seen immediately
following the first procedure (whereas the effect of IVIg may
take longer).
However, there are several indications where it is not yet
settled whether TPE is superior or IVIg is superior treatment
modality. To illustrate this point it would be pertinent to
quote this study on Guillain-Barré syndrome (GBS), which
divided patients into four categories based on treatment
received (IVIG, TPE, IVIG + TPE, or neither). Their finding
was that there is no association between PE after IVIG and
improved short-term outcomes of patients with Guillain-
Barré syndrome, but there was an association with an
increase in cost and duration of hospitalization. There was
no association between the timing of PE after IVIG and the
short-term outcome. Prospective studies are needed to
clarify whether the cost/benefit ratio favors the routine use of
this therapeutic approach.1 However, in GBS another study
claims potential benefit over IVIg in patients with axonal
involvement.2
References
1. Oczko-Walker M1,  Manousakis G,  Wang S,  et al. Plasma
exchange after initial intravenous immunoglobulin treatment
in Guillain-Barré syndrome: critical reassessment of
effectiveness and cost-efficiency. J Clin Neuromuscul Dis.
2010;12(2):55-61.
2. Dada MA1, Kaplan AA. Plasmapheresis treatment in Guillain-
Barré syndrome: potential benefit over IVIg in patients with
axonal involvement. Ther Apher Dial. 2004;8(5):409-12.
Therapeutic Erythrocytapheresis
and Leucapheresis
Nidhi Mehta
Apheresis is a general term describing a procedure in which
blood is removed from the body; a particular component of
blood is separated, and rest blood is then returned to body.
Therapeutic apheresis includes plasma exchange and
cytapheresis, which are generally tolerated by healthy donors.
However, many minor and a few major risks exist.
Plasma exchange (PE)
It is a procedure in which the plasma is separated from the
blood, discarded in total, and replaced with a substitution
fluid such as albumin or with donated plasma from a healthy
person. This is generally performed to remove toxins or
autoantibodies that have accumulated in the plasma.
Cytapheresis
Therapeutic cytapheresis removes cellular components from
blood, returning plasma.
The Apheresis Applications Committee of the American
Society for Apheresis periodically evaluates potential
indications for apheresis and categorizes them from I to IV
based on the available medical literature. The following are
some of the indications, and their categorization, from the
society’s 2013 guidelines.
Category I: Disorders for which apheresis is accepted as first-
line therapy, either as a primary standalone treatment or in
conjunction with other modes of treatment.
Category II: Disorders for which apheresis is accepted as
second-line therapy, either as a standalone treatment or in
conjunction with other modes of treatment.
Category III: Optimum role of apheresis therapy is not
established. Decision-making should be individualized.
Category IV: Disorders in which published evidence
demonstrates or suggests apheresis to be ineffective or
harmful. IRB approval is desirable if apheresis treatment is
undertaken in these circumstances.
37
Therapeutic Cytapheresis
It is most often used to remove defective RBCs and substitute
normal ones in patients with sickle cell anemia who have
the following conditions: acute chest syndrome, stroke,
pregnancy, or frequent, severe sickle cell crises. RBC
exchange achieves Hb S levels of < 30% without the risk of
increased viscosity that can occur because of increased Hct
with simple transfusion.
It may also be used to reduce severe thrombocytosis
or leukocytosis (cytoreduction) in acute leukemia or in
accelerated or blast crisis phase of chronic myelogenous
leukemia when there is risk of hemorrhage, thrombosis, or
pulmonary or cerebral complications of extreme leukocytosis
(leukostasis).
Erythrocytapheresis
A donor procedure in which the equivalent of 1 or 2 units of
RBCs are removed to produce RBCs for transfusion.
Therapeutic erythrocytapheresis (TEA) has been used in
different diseases such as polycythemia vera (PV), secondary
erythrocytosis or hemochromatosis as a process of the
less cumbersome but more expensive phlebotomy. TEA is
preferred in emergency conditions such as thrombocytosis
or in conditions such as porphyria cutanea tarda (PCT) or
erythropoietic porphyria when plasma exchange (PEX) is
often combined with TEA to reduce extracellular levels of
uroporphyrin which contribute to plasma hyper viscosity.
Leukocytapheresis
A procedure in which the WBCs are separated from the blood.
The cells may be discarded, as when used to decrease WBC
count in acute leukemia, or used for transfusion, as in the case
of granulocyte collection or the collection of hematopoietic
progenitor cells.
Therapeutic WBC removal (leukapheresis) can remove
kilograms of buffy coat in a few procedures, and it often
136  Section 1  Clinical Hemotherapy
relieves leukostasis. However, the reduction in WBC count
itself may be mild and only temporary.
Plateletapheresis
A donor procedure in which platelets are removed to produce
a platelet product for transfusion is called plateletpheresis.
Thrombocytapheresis
A therapeutic procedure in which platelets are removed and
discarded from a thrombocythemic patient. Cytapheresis is
effective in thrombocytosis because platelets are not replaced
as rapidly as WBCs. One or 2 procedures may reduce platelet
counts to safe levels.
Other uses of cytapheresis include collection of peripheral
blood stem cells for autologous or allogeneic bone marrow
reconstitution (an alternative to bone marrow transplantation)
and collection of lymphocytes for use in immune modulation
cancer therapy (adoptive immunotherapy).
Clinical Applications of Therapeutic
Erythrocytapheresis
• Primary and secondary polycythemia
• Sickle cell disease (SCD)
• For autologous and miscellaneous hematological appli­
cations
• For intraerythrocytic parasite infections
• Cerebral malaria
• ABO incompatible transfusions
• Prevention of Rh immunization after RhD+ transfusions
into RhD recipients
• CO intoxication
• Aniline poisoning
• AIHA
• Hb-Cheverly
• Hereditary hemochromatosis.
Therapeutic erythrocytapheresis (TE) has become a new
therapeutic modality, which potentially offers a more efficient
method to remove iron overload with fewer procedures.
Red cell exchange using a cell separator causes rapid
removal of parasites from the circulation of patients with
high parasite load with renal, pulmonary and cerebral
complications. It offers a rapid approach to treat acute, severe
cases of malaria.
Suggested Reading
1. Deshpande A, Kalgutkar S, Udani S. JAPI. 2003;51.
2. Journal of Clinical Apheresis. 2013;28:145-284.
3. Kozanoglu I, et al. Transfusion and Apheresis Science.
2007;36:305-12.
4. Therapeutic Apheresis and Dialysis. 14(2):230-33.
5. Valbonesi M, Bruni R. Transfusion Science. 2000;22:183-94.
Red Cell Exchange in
Clinical Practice
Joshua Daniel Jeyakumar, Prakash H Muddegowda, Jyothi B Lingegowda
Introduction
Red blood cell exchange (RCE) has been used over the years
for a large number of applications. The procedure involves
removal of patient red blood cells (RBCs) and replacing
with donor packed RBCs. It is specifically indicated in renal
damage secondary to erythrocytes like in sickle cell anemia
and severe malaria.1 In hemoglobinopathies, RCE can be
used as first line adjunct therapy or as primary therapy
especially in serious complications.2
The use of modern cell separators, the introduction of
leukodepletion in blood products, and the increasing safety
of red blood cell concentrates have made this treatment
modality very effective, comfortable and safe. It is frequently
used in developed countries since a long time. There are very
few centers performing RCEs in India.1
Red blood cell exchange Technique
Red blood cell exchange can be performed using automated or
manual cell separator with the inbuilt formulae or web-based
tools for calculating the red cell volume to be exchanged. In
the older patients, veno-venous access and in small children,
blood given back via port is usually followed. Well-trained
technicians are necessary to conduct the procedure. COBE
spectra and spectra optia are commonly used RCE separators.2
Indications of Red blood cell
exchange1
• Sickle cell disease (SCD)
• Protozoal infections of red blood cells (RBCs)
• Some types of poisoning like nitrobenzene
• Incompatible transfusion and hemolytic disease of new­
born
• Others like erythrocytosis, hereditary hemochromatosis,
refractory warm autoimmune hemolytic anemias and
porphyria, etc.
38
Sickle Cell Disease
Sickle cell disease is a group of inherited conditions identified
by the pre­sence of hemoglobin-S (HbS) or sickled RBCwhich
replaces a proportion of normal hemoglobin A. Sickled
RBC can lead to endothelial dysfunction, microvascular
complications, vasoocclusion, hyperviscosity and blood
sludging leading to severe vaso-occlusive crisis and
dysfunction of various organ systems.
Transfusion of normal (nonsickle) blood into patients
with sickle cell disease increases hematocrit (Hct) and
simultaneously (by dilution) lowers the fraction of cells that
contain HbS. By increasing the Hct, transfusion may also
reduce the erythropoietic drive due to anemic hypoxia and
thereby, decreased production of sickle hemoglobin will
occur. An RCE transfusion can be used to“replace” HbS by
HbA.3
Indications for Sickle Cell Disease4,5
The American Society for Apheresis (ASFA) delineates RCE
indications by the acuity of the clinical situation
For acute cases:
• Stroke—Category I
• Acute chest syndrome—Category II
• Priapism—Category III
• Multiorgan failure—Category III
• Splenic sequestration—Category III.
For chronic RBC exchange, the ASFA categories are:
• Stroke prophylaxis—Category II
• Vaso-occlusive crisis—Category III
• Preoperative management—Category II.
The target HbS level of below 30% may require repeated
exchanges. A single volume exchange will replace ~ 65% of the
patient’s cells; a double volume exchange will replace ~85%.
The aim is to exchange 0.5–1 blood volume in each exchange.
138  Section 1  Clinical Hemotherapy
The amount of blood to be exchanged (phlebotomized and
transfused) is computed and based on the following equation:
(Hctd – Hcti) × TBV
Exchange volume (mL) =
Hctrp – (Hcti + Hctd)/2
(Hctd, desired hematocrit; Hcti, initial hematocrit; Hctrp,
hematocrit of replacement cells (usually 0.7–0.8); TBV,
estimated total blood volume in mL).
The calculated amount of blood to be exchanged as based
on this equation is approximately 50–60 mL/kg of body
weight for an average adult. In the exchange transfusion
protocol used for pediatric patients, a volume of 10 mL/kg
of blood is phlebotomized and 15 mL/kg of blood infused
immediately.4–7
Major advantage of RCE in SCD has been its iron
neutrality, since the removed HbS has just as much iron as the
administered HbA. Treatment of patients with bone marrow
necrosis and multiorgan failure is mainly supportive.
Malaria
Mosquito-borne illness caused by infection of RBCs with
Plasmodium protozoa. In malaria, RCE offers a rapid ap­
proach to treat acute, severe cases of malaria. It is expected to
increase the oxygen carrying capacity of the blood along with
reduction of parasitic load, removal of toxic substances and
also reduction of microcirculatory sludging.4–7
The most commonly reported indications for RCE in
malaria is hyper parasitemia (>5–70% infected erythrocytes).
There have been several studies showing RCE as an adjunct
therapy in several Plasmodium falciparum malaria to be very
beneficial.8,9
WHO guidelines to provide RCE are: as an adjunct therapy-
level of parasitemia of >10% with or without disease, or failure
to improve after 24 hours of antimalarial chemotherapy.10
Babesiosis
Tick-borne, malaria-like illness caused by infection of RBCs
with Babesia protozoa. Characterized by flu like syndrome,
headache, chills, sweats, myalgia and arthralgia. Although
clinical trials are lacking, several case reports and case
series suggest that, given the absence of exo-erythrocytic
phase of infection, RCE combined with antibiotic therapy,
can be beneficial in severe cases of babesiosis with >5%
parasitemia.1,4,7
Nitrobenzene and other poisonings
Nitrobenzene poisoning is a rare but fatal condition. It causes
methemoglobinemia (Fe3+) and reduces oxygen carrying
capacity of red blood cells (RBC).
Nitrobenzene is commonly available in printing dyes,
ether and soap industry and is a suicidal poison. It initially
presents with cyanosis and progresses to dyspnea and
tachycardia. A high saturation of above 70% methemoglobin,
it can cause death. Diagnosis is based on persistent cyanosis
on oxygen therapy without improvement in observed
saturation of oxygen. Methylene blue is the antidote of choice
in nitro­benzene poisoning. Along with it, RCE and dextrose
are given for further support. RCE works by improving the
Oxygen carrying capacity of red blood cells.
Red blood cell exchange can also be used in poisonings
like carbon monoxide, nitrobenzene, arsenic and sodium
hypochlorite, etc.11–14
Hemolytic Disease of Newborn
Hemolytic disease of the newborn (HDN) is characterized
by the presence of IgG antibodies in maternal circulation,
which causes hemolysis in the fetus by crossing the placenta
and sensitizing red cells for destruction by macrophages in
the fetal spleen with consequent hyperbilirubinemia. RCE
removes circulating bilirubin, antibodies in plasma and
antibody-coated sensitized red blood cells (RBCs), replacing
them with RBCs compatible with maternal serum or neonates’
serum and providing albumin with new bilirubin site.15–17
Miscellaneous
In erythrocytosis conditions like polycythemia, hereditary
hemochromatosis, etc. compared to phlebotomy, RCE
is known to be superior to usual phlebotomy treatment
reducing risk of volumeic imbalances (also plasma containing
thrombocytes, coagulation factors and proteins) and is an
efficient, safe and well-tolerated method. The main problems
noticed were the cost, and is negated by the reduced risk
of thromboembolism and clear decrease in viscosity,
which makes the cost effectiveness balance sufficiently
positive.1,15,17–20
Conclusion
RCE use on a larger scale will reduce the cost of the procedure.
A well-trained team is necessary along with matching
equipment, which will make it a clinically very effective
procedure with low percentage of side effects that are not
dangerous to the patient. Despite these wide applications,
data concerning RCE is still limited. We are looking into
this interesting field of therapeutic apheresis for further
randomized trials and widening of applications.
References
1. Schwartz J, Winters JL, Padmanabhan A, Balogun RA, Delaney
M, Linenberger ML, et al. Guidelines on the use of therapeutic
apheresis in clinical practice—Evidence based approach from
the writing committee of the American society of apheresis:
The sixth special issue. J Clin Apher. 2013;28:145-284.
2. Red blood cell exchange and depletion procedures. (internet).
2012 (cited 2014 Jun 15). Available from: http://www.terumobct.
com/location/north-america/Documents/306670723B.pdf
 Chapter 38  Red Cell Exchange in Clinical Practice  139
3. Cho G, Hambleton IR. Regular long-term red blood cell
transfusions for managing chronic chest complications in
sickle cell disease. The Cochrane library. Published online:
8 Jan 2014. Available from: http://onlinelibrary.wiley.com/
doi/10.1002/14651858.CD008360.pub3/pdf
4. Draser E, Igbineweka N, Vasavda N, Free M, Awogbade M,
Allman M, et al. Blood transfusion usage among adults with
sickle cell disease—a single institution experience over ten
years. Br J Hemat. 2011;152;766-70.
5. Mahfoudhi E, Lecluse Y, Driss F, Abbes S, Flaujac C,
Garcon L. Red cell exchanges in sickle cell disease lead
to selective reduction of erythrocytes—derived blood
microparticles. Br J Hemat. 2012;156:545-7.
6. Swerdlow PS. Red cell exchange in sickle cell disease.
Hematology Am Soc Hematol Edu Program. 2006.pp.48-53.
7. Cheung ATW, Miller JW, Miguelino MG, To WJ, Lin X, Chen
PC, et al. Exchange transfusion therapy and its effects on real-
time microcirculation in pediatric sickle cell anemia patients:
An intravital microscopic study. J Peditr Hematol Oncol.
2012;34(3):169-74.
8. Udani S, Deshpande A, Kalgutkar S. Exchange transfusion for
severe malaria: A comparison of red cell exchange with whole
blood exchange. IJCCM. 2003;7(2):124-7.
9. Riddle MS, Jackson JL, Sanders JW, et al. Exchange transfusion
as an adjunct therapy in severe Plasmodium falciparum
malaria: A meta analysis. CID. 2002;34:1192-8.
10. Cabrera LS, Arroyo MF, Gonzalez FR, et al. Erythrocytapheresis
in the management of severe falciparum malaria. J Emerg
Trauma Shock. 2010;3(2):206.
11. Srivastava A, Chaturvedi A, Gupta SK, et al. Acute nitrobenzene
poisoning: case fatality and importance of methylene blue. Sri
Lankan Journal of Anaesthesiology. 2010;18(2):91-3.
12. Romanovsky A, Djogovic D, Chin D. A case of sodium chlorite
toxicity managed with concurrent renal replacement therapy
and red cell exchange. J Med Toxicol. 2013;9:67-70.
13. Saxena H, Saxena AP. Acute methaemoglobinemia due to
ingestion of nitrobenzene (paint solvent). Indian J Anaesth.
2010;54(2):160-2.
14. Adamski J, Hanna CA, Reddy VB, Litovsky S, Evans CA,
Marques MB. Multiorgan failure and bone marrow necrosis in
three adults with sickle cell beta thalassemia. Am J Hematol.
2012;87:621-4.
15. Stokley S. Guideline for exchange transfusions in children and
adolescents with sickle cell disease. (internet) 2011 (cited 2014
Jun15). Available from: https://www.nuh.nhs.uk/handlers/
downloads.ashx?id=48971.
16. Wilkinson K, Harris S, Gaur P, Haile A, Armour R, Teramura G,
et al. Molecular blood typing augments serologic testing and
allows for enhanced matching of red blood cells for transfusion.
in patients with sickle cell disease. Transfusion 2012;52:381-8.
17. Baron JM, Baron BW. Red cell exchange is not effective
for patients with sickle cell anemia and coexisting warm
autoantibody hemolysis. Blood Transfus. 2010;8:303-6.
18. Hackenberg LA, Staudinger T, Bojic A, Locker G, Leitner
GC, Graninger W, et al. Automated red cell exchange as an
adjunctive treatment for severe Plasmodium falciparum
malaria at the Vienna General Hospital in Austria: a retro­
spective cohort study. Malar J. 2012;11:158.
19. Deshpande A, Kalgutkar S, Udani S. Red cell exchange using
cell separator (Therapeutic eryththrocytapheresis) in two
children with acute severe malaria. JAPI. 2003;51:926-6.
20. Watanaboonyongcharoen P, Park YA, Poisson JL, et al. Rapid
increase in parasitemia following red cell exchange for malaria.
J Clin Apher. 2011;26(6):315-9.
 Chronically Transfused Patients
Transfusion Practice in
Thalassemia
Gagandeep Kaur
Introduction
Thalassemia is considered as the most common monogenic
disorder worldwide with a particularly high frequency in a
broad belt, extending from Mediterranean region, Africa, the
Middle East, the Indian subcontinent, Myanmar and South-
east Asia. About 10% of total world thalassemia patients
belong to the Indian subcontinent, among them 3–4% are
carriers. These disorders occur due to decreased or absent
synthesis of the globin chains. Alpha and beta thalassemias
are two most important forms of the disorder. Clinically the
most severe form of the disorder is classified as thalassemia
major and is transfusion dependent, less severe but clinically
symptomatic disease is called thalassemia intermedia and
the generally asymptomatic carrier state is the thalassemia
trait.
Pathophysiology
In thalassemia major, the genetic defect results in absence
of normal globin chain synthesis with resultant excess of the
other unaffected globin chains. The excess chains precipitate
in the erythroid precursors leading to intramedullary death
and ineffective erythropoiesis.1 The damage is much more
with excess alpha chains hence ineffective erythropoiesis is
markedly increased in beta thalassemia major. The resultant
anemia leads to increased erythropoietin production; hence
there is stimulation of erythropoiesis both medullary and
extramedullary. Increase in plasma volume as a result of
shunting through expanded bone marrow and progressive
splenomegaly aggravate the anemia. Bone marrow expansion
also results in characteristic deformities of the skull and face.
There is increased gastrointestinal iron absorption due to
paradoxical hepcidin suppression from dyserythropoiesis,
which leads to progressive iron deposition in tissues and a
variety of growth, metabolic and organ abnormalities.
39
Transfusion therapy
The current management of thalassemia is based on regular
transfusions and effective chelation therapy. The goals of
transfusion therapy are the correction of anemia, suppression
of erythropoiesis and inhibition of gastrointestinal iron
absorption, which occurs in transfused patients as a conse­
quence of increased, although ineffective erythropoiesis.
Whom to transfuse
The decision to start transfusion in patients with a confirmed
diagnosis of thalassemia should be based on the presence of
severe anemia (Hb < 7 g/dL on 2 occasions, more than two
weeks apart, excluding all other contributory causes such
as infections). However, even in patients with hemoglobin
> 7 g/dL, other factors should be considered, including age at
presentation of first symptoms, facial changes, poor growth,
evidence of bony expansion and increasing splenomegaly.
When possible, the decision to start regular transfusions
should not be delayed until after the third year, as the risk
of developing multiple red cell antibodies increases, with
subsequent difficulty in finding suitable blood units.
Compatibility testing
The usual transfusion policy is to perform ABO and Rh(D)
typing of donors and patients and subsequent compatibility
testing. However, there are many minor but clinically signi­
ficant blood groups which can lead to alloimmunization
in these patients. Once the alloantibodies develop, finding
compatible units may become difficult. Following steps
should be followed before starting the transfusion therapy:
• Extended red cell antigen typing of the patient before
starting transfusion: The patient should be typed for

common antigens of the Rh, Kell, Kidd and Duffy systems
to prevent RBC alloimmunization.
• Ethnically related donors: Homogeneity between donor
and patient populations leads to a lower prevalence of
alloimmunization. Some workers emphasize on recruit­
ment of phenotypically matched donors.
• Antibody screening in the patient prior to each transfu-
sion.
• Hepatitis B vaccination.
• Human leukocyte antigen (HLA) typing of siblings
and patients should be done. Transfusions from 1st
degree relatives should be avoided if bone marrow
transplantation (BMT) is required in future.
• Family study for genetic counseling.
Transfusion regimen
The superiority of regular repeated transfusions, as compared to
transfusions only for symptomatic anemia, was first recognized
by Orsini in France and later by Wolman and Piomelli in US,
who suggested a transfusion program aimed at monitoring a
basal Hb level sufficient to eliminate hypoxia.2 Several different
regimens have been proposed over the years, but at present the
majority of centers choose to transfuse at a pretransfusion Hb
level of 9–10 g/dL, and to reach a post-transfusion level of 13–
14 g/dL. This prevents growth impairment, organ damage and
bone deformities, allowing normal activity and quality of life,
and is associated with relatively low rates of blood requirement
and of iron accumulation. Post-transfusion hemoglobin
should not exceed 15 g/dL to prevent thromboembolism.
In untreated beta thalassemia major, the erythropoiesis
exceeds 10 times the normal leading to bony abnormalities.
Gastrointestinal iron absorption increases over and above the
body iron losses when the erythroid activity exceeds 5 times
normal. There is almost complete suppression of endogenous
erythropoiesis with hyper transfusion and super transfusion
regimen; however with moderate transfusion regimen the
erythroid activity exceeds 2.4 times normal. This is below the
critical threshold of five times normal.3
Transfusion interval
The frequency of transfusion is usually every three to four
weeks. The amount of blood to be transfused depends on
several factors including the weight of the patient, and the
target increase in Hb level. Appropriate graphs and formulae
to calculate the amount of blood to be transfused are
available. In general, the amount of transfused RBC should
not exceed 15–20 mL/kg/day, infused at a maximum rate of
5 mL/kg/hour to avoid a fast increase in blood volume.
Characteristics of blood products
for transfusion
Careful selection of healthy voluntary donors is a prerequisite
for obtaining safe blood units for patients with thalassemia.
Chapter 39  Transfusion Practice in Thalassemia  141
Patients with thalassemia suffer from chronic anemia and
hence require red blood cells. Despite increasingly sophisti­
cated blood additives, RBCs suffer from ex vivo storage and
undergo biochemical, structural, enzymatic, morphological
and functional deterioration.4 Although many of these changes
are reversible following transfusion, the current practice is to
transfuse red cells in appropriate additive solutions for less
than one week and hence as fresh as possible.5,6
Modified Packed RBCs
Leukoreduced
To avoid transfusion reactions from antiplatelet antibodies
and antileukocyte antibodies, patients should receive
leukoreduced packed red cells. Removal of leukocytes and
platelets is obtained by filtration of whole blood. Reduction
to 1 × 106 or fewer leukocytes per unit is considered. The
critical threshold for preventing adverse reactions attributed
to contaminated leukocytes.7
Washed Packed RBCs
In rare patients sensitized to plasma proteins, washed red
cells may be beneficial.
Neocytes
These are young red cells with low specific gravity and can
survive in the circulation longer than older cells. They can
be separated from donor units’ ex-vivo using cell washers
or using continuous flow cell separators. Usually 50% of
the original red cell volume is recovered as neocytes. The
clinical benefits of neocyte transfusions are (a) increase of
transfusion interval, (b) reduction in packed RBC transfusion
requirements and (c) reduction in transfused iron by 15%
per year per patient. However, two disadvantages are: (a)
considerably increased donor exposure and (b) increase cost
of processing.
Effectiveness of transfusion therapy
To evaluate the effectiveness of transfusion therapy, some
indices should be recorded at each transfusion, including
pre- and post-transfusion Hb, amount and hematocrit
(Hct) of the unit, daily Hb fall, and interval between
transfusions. These measurements enable two important
parameters to be calculated: red cell requirement and iron
intake. Dedicated computerized programs are available to
monitor transfused thalassemia patients. If the annual red
cell requirement exceeds 1.5 times that of splenectomized
patients, splenectomy should be considered, provided that
other reasons for increased consumption, such as hemolytic
reactions have been excluded. For patients maintaining a
pretransfusion Hb of 9.5 g/dL, the increase in transfusion
requirement is represented by a consumption of more than
142  Section 1  Clinical Hemotherapy
200 mL of RBC/kg/year (assuming that the Hct of the unit of
red cells is 75%).
Problem of thalassemia transfusion
programs is developing countries
• Fragmented management of blood transfusion services-
lack of quality check in testing.
• Inadequate voluntary blood donation.
• Absence of component preparation facilities in most of the
blood centers hence packed RBCs may not be available.
• Absence of extended phenotype testing in patients and
donors.
• No screening facilities for irregular antibodies.
• Nucleic acid testing for viral markers not in place to reduce
window period transmission.
• Unaffordability of leukofilters.
• Chelation compliance and poor affordability.
Conclusion
Efforts of the health care providers should be directed to
improve the early diagnosis and management of β-thalas­
semia so that disease complications can be prevented. Pre-
implantation genetic diagnosis is an option that has become
widely accepted to prevent severe β-thalassemia.
References
1. Weatherall DJ, Clegg JB. The Thalassemia Syndromes. Fourth
Edition. Blackwell Science. 2001.
2. Piomelli S, Danoff SJ, Becker MH, et al. Prevention of bone
malformations and cardiomegaly Cooley’s anemia by early
hypertransfusion regimen. Ann NY Acad Sci. 1969;165:427-36.
3. Cazzola M, Borgna-Pignatti C, Locatelli F, et al. A moderate
transfusion regimen may reduce iron loading in beta-
thalassemia major without producing excessive expansion of
erythropoiesis. Transfusion 1997;37:135-40.
4. Dzik W. Fresh blood for everyone? Balancing availability and
quality of stored RBCs. Transfus Med. 2008;18:260-5.
5. Thalassemia International Federation (T.I.F.). Guidelines for
the clinical management of thalassemia. 2008.
6. Goss C, Giardina P, Degtyaryova D, Kleinert D, et al . Red cell
transfusions for thalassaemia: results of survey assessing
current practice and proposal of evidence based guidelines.
Transfusion. 2014;54:1773-81.
7. Norfolk DR, Williamson LM. Leucodepletion of blood products
by filtration. Blood Rev. 1995;9:7-14.
Pediatrician and β-Thalassemia
Parveen Mittal
INTRODUCTION
India is an ethnically diverse country with an approximate
population of 1.5 billion. The frequency of beta-thalassemia
trait has variously been reported from <1 to 17% and an
average of 3.3%.
The word “thalassemia” comes from the Greek word
“thalassa” (sea) because of the high prevalence of the disease
in the countries bordering the Mediterranean Sea. Beta-
thalassemia had been traditionally prevalent in and confined
to the Mediterranean basin, Middle East, North India,
Southeast Asia and the Indochina Peninsula.1-3
It has been estimated that the prevalence of pathological
hemoglobinopathies in India is 1.2/1,000 live births, and with
approximately 27 million births per year this would suggest
the annual birth of 32,400 babies with a serious hemoglobin
disorder. Within this overall disease classification a 1989 WHO
working group on guidelines for the control of hemoglobin
disorders estimated a 3.9% carrier frequency for β-thalassemia
in India, encompassing all types of β-thalassemia trait.4–6
Beta-thalassemia is caused by the reduced synthesis
of beta-globin chains, which leaves an erythrocyte excess
of unopposed alpha-chains; the resulting ineffective
erythropoiesis leads to chronic hemolytic anemia.7
Three clinical forms of beta-thalassemia are distinguished,
depending on clinical severity:
1. Thalassemia major: The typical phenotype arising either
from homozygous or from compound heterozygous
defects. This kind of anemia is severe, develops during the
first year of life, and requires life-long transfusion therapy
for survival.
2. Thalassemia intermedia: Accounts for up to 25% of cases
and encompasses a wide variety of molecular defects,
including homozygous, compound heterozygous, and
double heterozygous ones. The phenotypic spectrum
extends between the clinically severe thalassemia major
on one end and asymptomatic thalassemia carrier state
on the other end. Anemia in thalassemia intermedia is
generally milder than that of the major form and not
40
transfusion dependent, whereas the disease has a later
clinical onset and is associated with longer survival
compared with thalassemia major.
• Thalassemia minor: mild asymptomatic disease.8
Recent advances in detection and therapeutic management
of thalassemia has resulted in substantial improvement of
patients’ survival.
ROAD MAP
• Diagnosing thalassemia
• Educating and counseling parents
• Initiation and monitoring of treatment
• Follow-up
• Preventive strategy.
Diagnosing Thalassemia
There are only a few causes for anemia in first year of
life, thalassemia being one of the major causes among
them. Punjab being a belt of increased incidence of beta
thalassemia, we should have a high degree of suspicion in
diagnosing the disease. The delay in diagnosing and treating
the disease portends multisystem complications leading to
poor quality of life and even mortality.
Patients with the beta thalassemia trait generally have no
unusual physical findings. In patients with beta thalassemia
major, the physical findings are related to severe anemia,
ineffective erythropoiesis, extramedullary hematopoiesis,
and iron overload resulting from transfusion and increased
iron absorption. The skin may show pallor from anemia and
jaundice from hyperbilirubinemia, and the skull and other
bones may be deformed secondary to erythroid hyperplasia
with intramedullary expansion and cortical bone thinning.
Heart examination may reveal findings of cardiac failure and
arrhythmia, related to either severe anemia or iron overload.
Abdominal examination may reveal changes in the liver,
gallbladder, and spleen. Hepatomegaly related to significant
extramedullary hematopoiesis typically is observed.
144  Section 1  Clinical Hemotherapy
Patients who have received blood transfusions may have between 9.5 and 10.5 g/dl, thus improving his or her sense
hepatomegaly or chronic hepatitis due to iron overload. The of well-being while simultaneously suppressing enhanced
gallbladder may contain bilirubin stones formed as a result of erythropoiesis. But blood transfusion brings along a wagon
the patient’s lifelong hemolytic state. Splenomegaly typically of demerits such as–blood born infections and iron overload,
is observed as part of the extramedullary hematopoiesis which are discussion in detail later.
or as a hypertrophic response related to the extravascular
hemolysis. Iron overload may also cause endocrine Chelation Therapy
dysfunction, especially affecting the pancreas, testes, and
thyroid. Transfusion-associated viral hepatitis resulting in Chronic blood transfusion may lead to iron overload if not
cirrhosis or portal hypertension also may be seen. proper chelation is instituted. Increased amount of iron
One of the most common dilemmas faced by a may lead to multisystem involvement in form of secondary
pediatrician is to differentiate thalassemia from its common diabetes mellitus, hypothyroidism, hypoparathyroidism,
hematological mimickers, iron deficiency anemia being one hypogonadotropic gonadism, congestive heart failure and
of the most common. A major proportion of children in our cardiac arrhythmias.
country are malnourished with a resultant high incidence of
nutritional anemia. There should exist an in hospital protocol Deferoxamine
to differentiate between these similar yet different conditions.
Hematological and biochemical parameters useful in • it may be administered as a subcutaneous infusion over
differentiating these two conditions have been summed up in 10–12 hours 5–6 days a week.
the table given below. • Side effects—ototoxicity with high frequency hearing
loss, retinal changes, peripheral neuropathy and bone
Variable Beta-thalassemia Iron deficiency anemia dysplasia with truncal shortening.
Mentzer index* < 13 > 13
Red cell Normal Increased
Deferasirox
distribution width • The recommended initial oral daily dose is 20 mg/kg
Serum ferritin Normal or increased Decreased body weight. An initial daily dose of 30 mg/kg may be
Serum iron Normal to high <30
considered for patients who require reduction of elevated
body iron levels and who are also receiving more than
Total iron-binding Normal > 360 14 ml/kg/month of packed red blood cell.
capacity (TIBC)
• Side effects—abdominal pain, diarrhea, dizziness, earache
Percent transferrin 30–80 < 10 or pain in the ear, nausea, voice changes, vomiting.
saturation
Hb electrophoresis Increased HbA2, HbF Normal Folic Acid
* Mentzer index = mean corpuscular volume (MCV, in fL) divided by the red
blood cell count (RBC, in Millions per microliter) 200–500 mcg/day may be supplemented so as to tide over the
high turnover state.
MANAGEMENT
Stem Cell Transplantation
Management of beta thalassemic children has to have a multi
prong strategy. Beta-thalassemia can only be cured by allogenic hemato­
poietic stem cell transplantation (HSCT). HSCT treatment
of thalassemia has substantially improved over the last two
Caretaker Counselling decades, with advancements in preventive strategies, control
Education of the care takers occupies a major cog in the of transplant-related complications, and preparative regimens.
wheel. Nature of the disease, its chronicity, treatment options
and genetic counselling should be made available for the Splenectomy
parents even at the first sitting.
It may be indicated for patients with thalassemia intermedia
who have a falling steady-state hemoglobin level and for
Blood Transfusion transfused patients with a rising transfusion requirement.
Anemia in thalassemia is due to hemolytic process existing
in the system to clear out the damaged RBCs. This leads to
myriad of clinical symptoms which brings a child to our
Complications of Thalassemia
attention. Decision for transfusion should be individualized Complications associated with beta-thalassemia, aside from
with a goal of maintaining pretransfusion hemoglobin the aforementioned anemia, are as follows:

• Extramedullary hematopoiesis—pathogenic bone frac-
tures, spinal cord compression.
• Growth failure—higher metabolic need, hepatospleno-
megaly interfering with nutritional intake, chelation ther-
apy, endocrinopathy.
• Iron overload
– Hyperglycemia
– Hypocalcemia due to hypoparathyroidism
– Hypothyroidism
– Hypogonadotropic hypogonadism.
• Transfusion-associated infections.
• Cardiac arrhythmias and failure.
• Cholelithiasis.
FOLLOW-UP
Transfusion requirement in every child varies and the same
should be individualized. The consulting pediatrician should
device a follow-up algorithm incorporating surveillance over
the major complications of thalassemia and its treatment so
as to identify the complications early and institute effective
management.
The follow-up algorithm should include:
• Anthropometric assessment including plotting growth
charts
• Hemodynamic parameters
• Echocardiography
• Effort tolerance
• Nutrition
• Viral markers
• Periodical monitoring of thyroid, parathyroid and liver
functions
• Glycemic status
• Status of iron overload—serum ferritin, iron studies, liver
biopsy, T2 weighted MRI of liver and heart
• Monitoring for adverse effects of chelation therapy.
PROGNOSIS
Individuals with thalassemia minor (thalassemia trait) usually
have mild, asymptomatic microcytic anemia. This state does
not result in mortality or significant morbidity. The prognosis
of patients with thalassemia major is highly dependent on the
patient’s adherence to long-term treatment programs, namely
the hypertransfusion program and lifelong iron chelation.
Allogenic bone marrow transplantation may be curative.
Morbidity and Mortality
The major causes of morbidity and mortality in beta-
thalassemia are anemia and iron overload. The severe
anemia resulting from this disease, if untreated, can result
in high-output cardiac failure; the intramedullary erythroid
expansion may result in associated skeletal changes such as
cortical bone thinning. Increased iron deposition resulting
Chapter 40  Pediatrician and β-Thalassemia  145
from lifelong transfusions and enhanced iron absorption
results in secondary iron overload.
PREVENTION
Effective prevention programs for thalassemia have been
demonstrated in many European countries where the
numbers of carriers of abnormal genes are high. Premarital
screening for thalassemia should be made a standard
practice. Most at-risk couples are identified early for prenatal
diagnosis in the first pregnancy and genetic counseling may
be offered to these individuals to have healthy offspring.
Screening program need to be supported by public education
and regulatory structures empowering individuals to make
informed decisions and to ensure that people are protected
against discrimination as a result of their test results.
Thalassemia carriers may be detected mainly through the
expanded family study of cases with thalassemic diseases.
Local data are still not available due to inherent difficulties
in the diagnosis of thalassemia traits. However, laboratory
progress during the last few years has enabled us to undertake
mass screening for beta-thalassemia and Hb E carriers, with
accurate diagnosis using automatic high performance liquid
chromatography (HPLC).
In our community reliable estimates for the prevalence of
thalassemia genes are not available at present, and diagnoses
of the diseases and management of the patients are still far
from satisfactory. Thus, the patients and families suffer
miserably due to ignorance about the disease, misunder­
standing and malpractices. To improve this situation will
require proper education of health personnel, not only the
physicians but downstream to the auxiliaries working in the
communities. Genetic counseling, at least for families already
afflicted by the diseases or in those who request it, should
be provided. This requires accurate diagnoses and a good
understanding of the different disorders. While conventional
therapy involving blood transfusions and iron chelation
are intolerably expensive, prenatal diagnosis and selective
abortion are more economical and cost-effective. However,
these should not be carried out indiscriminately.
PRENATAL DIAGNOSIS
• Chorionic villous sampling and DNA analysis in the first
trimester.
• Molecular analysis–β-thalassemia is extremely hetero­
geneous with more than 200 mutations described
worldwide.9 In India, about 64 mutations have been
characterized by studies done at different centers10-13
• Fetal blood analysis in the second trimester–In India,
second trimester diagnosis is still done as many couples
at risk are identified late during pregnancy. Fetal blood
sampling is done by cordocentesis at 18–20 week
gestation and after confirming that there is no maternal
contamination in the fetal sample by fetal cell staining
146  Section 1  Clinical Hemotherapy
using the Kleihauer-Betke method, it is analysed by HPLC
on the variant hemoglobin testing system.
CONCLUSION
In a resource poor setting like that of rural Punjab, pediatricians
can make a difference in by forming a local protocol for
diagnosis and management of thalassemia. Beta- thalassemia
being a genetic disorder clusters in families. Management
should be started even before the disease start to exist. The
current era of giant strides in the fields of medicine allows us
to predict, prevent and eradicated many diseases. Let us heal
the world, make it a better place, for the entire human race.
REFERENCEs
1. Kremastinos DT, Tsiapras DP, Kostopoulou AG, Hamodraka ES,
Chaidaroglou AS, Kapsali ED. NT-proBNP levels and diastolic
dysfunction in beta-thalassaemia major patients. Eur J Heart
Fail. 2007;9:531-6.
2. Modell B, Khan M, Darlison M. Survival in beta-thalassaemia
major in the UK: data from the UK Thalassaemia Register.
Lancet. 2000;355:2051-2.
3. Olivieri NF, Nathan DG, MacMillan JH, Wayne AS, Liu PP,
McGee A, Martin M, Koren G, Cohen AR. Survival in medically
treated patients with homozygous beta-thalassemia. N Engl J
Med. 1994;331:574-8.
4. Engle MA, Erlandson M, Smith CH. Late cardiac complications
of chronic, refractory anemia with hemochromatosis.
Circulation. 1964;30:698-705.
5. Borgna-Pignatti C, Rugolotto S, De Stefano P, Piga A, Di
Gregorio F, Gamberini MR, Sabato V, Melevendi C, Cappellini
MD, Verlato G. Survival and disease complications in
thalassemia major. Ann N Y Acad Sci. 1998;850:227-31.
6. World population data sheet. Washington DC: Population
Reference Bureau; 2009.
7. Kremastinos DT, Toutouzas PK, Vyssoulis GP, Venetis CA,
Avgoustakis DG. Iron overload and left ventricular performance
in beta thalassemia. Acta Cardiol. 1984;39:29-40.
8. Kremastinos DT, Tsiapras DP, Tsetsos GA, Rentoukas EI,
Vretou HP, Toutouzas PK. Left ventricular diastolic Doppler
characteristics in beta thalassemia major. Circulation.
1993;88:1127-35.
9. Globin Gene Server: http://globin.cse.psu.edu/, accessed on
October 5, 2010).
10. Colah R, Gorakshakar A, Nadkarni A, Phanasgaonkar S, Surve
R, Sawant P, et al. Regional heterogeneity of beta thalassemia
mutations in the multi ethnic Indian population. Blood Cells
Mol Dis. 2009;42:241-6.
11. Sinha S, Black ML, Agarwal S, Colah R, Das R, Ryan K, et al.
Profiling β-thalassemia mutations in India at state and regional
levels: implications for genetic education, screening and
counseling programmes. Hugo J. 2009;3:51-62.
12. Edison ES, Shaji RV, Devi SG, Moses A, Viswabandhya A,
Mathews V, et al. Analysis of β-globin mutations in the Indian
population: presence of rare and novel mutations and region
wise heterogeneity. Clin Genet. 2008;73:331-7.
13. Kumar R, Tamhankar PM, Panigrahi I, Dalal A, Agarwal S.
A novel β-globin mutation (HBB; c: 107 A>G; or codon 35 β
(A to G) at alpha-beta chain interfaces. Ann Hematol. 2009;88:
1269-71).
Iron Overload in Chronically
Transfused Patients: Diagnosis
and Treatment
Praveen C Sobti, Shruti Kakkar
41
Regular blood transfusion is required for management of a hepatitis and depressed in vitamin C deficiency. Studies have
variety of hematological conditions (Table 1). Each unit of demonstrated a significantly lower risk of cardiac failure and
packed cells transfused adds 200 mg of iron to the body’s iron death if serum ferritin is kept below 2500 µg/L over a decade.
pool. Moreover, many of these patients also have increased A serum ferritin of less than 1000 µg/L, is associated with
absorption of iron from the gut due to chronic anemia. longer survival and reduced risk of cardiac complication and
There is no physiological mechanism for removing this hypogonadism.
excessive iron from the body. Without iron chelation, iron Serum ferritin is measured serially every 3–6 months
gets deposited in liver, heart and endocrine organs leading interval in patients with thalassemia major and chelation
to complications. Adequate iron chelation is the cornerstone therapy tailored accordingly. A trend in serum ferritin is
of management of these disorders. The goal of iron chelation more important than a single value while deciding upon the
has shifted from treating iron overload to prevention of iron chelation regimen.
deposition in tissues.
Liver Iron Concentration
Assessment of Iron Overload Liver iron concentration is the reference standard for
estimating the total body iron.
Regular and accurate assessment of the iron overload forms
Liver iron concentration can be measured by chemical
the basis of an effective iron chelation regimen.
determination on a liver biopsy specimen or by noninvasive
methods like superconducting quantum interference device
Serum Ferritin (SQUID) and MRI.
Serum ferritin is a readily available, inexpensive, indirect Liver biopsy: Liver biopsy is considered the gold standard
measure of body iron stores. Serum ferritin has been shown for estimating the iron overload as it is a direct measure of
to correlate with the body iron stores in thalassemia major but LIC and also gives additional information on liver histology.
underestimate the iron loading in thalassemia intermedia. Normal LIC values are up to 1.8 mg/g dry weight of the liver.
Serum ferritin can be falsely elevated in inflammation, Values of up to 7 mg/g dry weight have been seen in normal
population without any adverse effects. High liver iron
content (LIC > 15–20 mg/g dry weight) has been associated
with worsening prognosis, liver function abnormalities
Table 1: Hematological conditions which may require repeated and progression to liver fibrosis. Liver biopsy is an invasive
transfusions and iron chelation
procedure but the rate of complications in experienced hands
Congenital Acquired has been reported to be less than 0.5%. Fallacious values can
Thalassemia major Myelodysplastic syndrome be obtained due to inadequate sample size (< 2.5 cm core
Thalassemia intermedia Aplastic anemia size) or uneven distribution of iron especially in presence of
cirrhosis.
Sickle cell disease Paroxysmal nocturnal
hemoglobinuria
SQUID
Sideroblastic anemia
Liver iron content (LIC) can be measured accurately by mag­
Pure red cell aplasia
netic biosusceptometry using a superconducting quantum
Fanconi’s anemia interference device (SQUID). Only 4 such machines are
148  Section 1  Clinical Hemotherapy
available worldwide, limited availability, high maintenance
cost and requirement for dedicated trained staff limits its
widespread utilization.
MRI
The concept of magnetic resonance imaging (MRI) assessment
of iron overload by MRI came up in 1980’s but only recently
the advances in the MRI techniques have made it possible to
use MRI for widespread assessment of iron overload.
The underlying principle is that the organs containing
iron darken more rapidly than normal tissues on a MRI. The
echo or observation time taken by a tissue to become twice as
dark is represented as T2*. The rate of darkening of a tissue is
expressed as R2*. T2* is inversely related to R2* and vice versa
(T2* = 1000/R2*). MRI machines can also measure T2 and R2,
but this is technically more demanding and slow.
MRI values are affected by the tissue water concentration.
An increase in R2 and R2* is found in dehydrated states due
to concentration effect whereas opposite occurs in fluid
overload.
MRI Assessment of Liver Iron
Liver iron content (LIC) can be calculated from R2 and
R2*. R2* is easy to obtain but requires a special software for
generating iron estimates (Table 2). Both R2 and R2* can be
converted to biopsy equivalents using calibration curves.
The maximum liver iron concentration that can be reliably
obtained from a MRI is between 30 and 40 mg/g dry weight
on a 1.5 Tesla machine.
MRI Assessment of Cardiac Iron
Cardiac T2*or R2* can be measured using the same scanner
and software as liver R2* but is more labor intensive. It can
also give additional information regarding the chamber size
and function at the same time. Cardiac iron content can be
calculated from T2* as
Cardiac Fe = 45(T2*)–1.22
Cardiac T2* > 20 milliseconds is considered normal and
corresponds to a cardiac iron content of 1.6 mg/g dry weight.
Cardiac T2* of < 10 ms is considered severe iron overload
and these patients are at high risk of developing heart failure
(Table 3). Studies have demonstrated that 50% of patients
with cardiac T2* < 6 ms develop heart failure within a year.
Table 2: Liver T* and iron overload
Grading Hepatic T2*(ms)
None > 6.3
Mild 2.7–6.3
Moderate 1.4–2.7
Severe < 1.4
Table 3: Cardiac T2* and iron overload
Grading Myocardial T2*(ms)
None > 20
Mild 12–20
Moderate 8–12
Severe <8
Table 4: Pancreatic R2* and iron overload
Pancreatic R2* Iron overload
< 30 Hz Normal
30–100 Hz Mild
100–400 Hz Moderate
> 400 Hz Severe
MRI Assessment of Pancreatic Iron Overload
Pancreatic R2* is currently not being used in routine clinical
practice but gives complimentary information regarding iron
overload in addition to liver and heart estimates. Pancreatic
iron overload is a reflection of chronic NTBI exposure.
Pancreatic R2* is a better predictor of cardiac iron overload
than liver iron as the kinetics of pancreatic iron loading
and overloading are intermediate between heart and liver.
Correlation between R2* and pancreatic siderosis is shown
in Table 4. Longitudinal studies have demonstrated that
a pancreatic R2* < 100 Hz rules out cardiac iron overload
(negative predictive value 95%). Some authors have used a
staged approach where patients with pancreatic R2* of < 100 Hz
are followed with abdominal MRI only but this approach
needs to be validated. Pancreatic R2* > 100 Hz is also predictive
of impaired glucose tolerance. Pancreatic MRI is difficult
in older patients with thalassemia who have undergone
splenectomy due to lobular atrophy and fat stranding and can
give inaccurate assessment of iron overload in these patients.
MRI in other Iron Overload Conditions
MRI has been extensively studied in thalassemia major
but is also required in other chronically transfused states
like Diamond Blackfan anemia, thalassemia intermedia,
myelodysplastic syndromes and sickle cell anemia. Cardiac
iron overload is higher in Diamond Blackfan anemia and
least common in thalassemia intermedia.
Iron Chelation
The primary objective of chelation therapy is to keep body
iron within safe limits. The labile plasma iron constitutes the
chelatable iron pool as iron cannot be directly removed from
ferritin or hemosiderin. This accounts for the slow removal of
 Chapter 41  Iron Overload in Chronically Transfused Patients: Diagnosis and Treatment  149

iron once it is accumulated in the body. What constitutes the
safe iron levels depends upon the diagnosis and the chelation
being used.
When to Start Chelation
Iron chelation should be initiated as soon as transfusions have
caused enough iron accumulation to cause tissue damage.
Traditionally, with desferrioxamine iron chelation is started
when the following criteria are met due to fear of toxicity.
• Age of the child ≥ 2 years
• After first 10–20 transfusions
• Serum ferritin > 1000 µg/L
• LIC > 7 mg/g dry weight.
With the availability of newer oral iron chelators, how early
chelation can be initiated remains to be seen. The primary
goal should be prevention of iron loading of hepatocytes thus
preventing the secondary distribution to endocrine organs
and heart.
Desferrioxamine
Desferrioxamine (DFO) is the first iron chelator discovered
in 1962. This hexadentate chelator is obtained from
Strepyomyces Pilosus. Due to its high molecular weight, the
oral bioavailability is negligible. It has a short half-life of 20
min and the iron free drug is rapidly metabolized in liver.
The iron is excreted in both urine and feces (Table 5). The
urinary iron is derived from breakdown of red blood cells
in macrophages whereas the fecal iron is derived from iron
chelated within the liver.
Effect on Serum Ferritin
Desferrioxamine (DFO) lead decrease in serum ferritin
has been seen in clinical experience over 4 decades now.
Prospective studies of changes in serum ferritin in thalassemia
major patients have shown a dose dependent fall in serum
ferritin with DFO. A mean daily dose of 42 mg/kg lead to a
decrease in serum ferritin of 364 µg/L over a year compared
to a fall of 1000 µg/L with a mean daily dose of 51 mg/kg.
A trend in serum ferritin gives information on compliance
rather than iron content of body at a given time.
Effect on Liver Iron
DFO is effective in controlling liver iron if administered in
adequate doses at least 5 times a week. Randomized trials
have demonstrated that the effect of DFO on iron balance
depends on the rate of transfusional iron loading and the
dose given. A negative iron balance could be achieved in 75%
patients receiving DFO at 39–45 mg/kg/d, five days a week, if
transfusional iron loading rate was between 0.3 and 0.5 mg/
kg/d. With higher transfusional iron loading rates (> 0.5 mg/
kg/d), negative iron balance could be obtained in less than
50% patients at same dose, but increased to 86% at a dose of
50 mg/kg/d.
Effect on Cardiac Iron
Subcutaneous DFO changed the clinical progression in
thalassemia major. Before the introduction of DFO, most
patients of thalassemia major succumbed to iron induced
cardiac failure in second decade of life. Treatment with
continuous intravenous DFO has been shown to reverse the
symptomatic cardiac failure.
Continuous intravenous DFO has also been shown to
reduce the cardiac iron overloaded in severely iron loaded
hearts (T2* < 6 ms). It lead to an average increase in T2* of
about 3 ms/year. It would take several years for the cardiac
T2* to normalize at this linear rate.

Table 5: Comparison of 3 iron chelators

DFO DFP DFX
Mol wt 560 139 373
Chelator: iron Hexadentate Bidentate Tridentate
1:1 3:1 2:1
Route of Subcutaneous/ Oral Oral
administration Intravenous
Plasma half life 20 min 1.5 hrs 9–11 hrs
Dose (mg/kg/d) 20–40 75–100 20-40
Iron excretion Urine, fecal Urine Fecal
Side effects Local reactions, auditory, retinal and bony Gastrointestinal, Gastrointestinal, increase in
abnormalities, growth retardation Agranulocytosis, serum creatinine, skin rash
Arthropathy, zinc deficiency
Advantages Longest clinical experience Good for cardiac siderosis Once daily administration
Disadvantages Parenteral administration Weekly monitoring of blood Cost
counts
150  Section 1  Clinical Hemotherapy
Other Effects
Regular subcutaneous DFO infusions started before the age
of 10 years has also been shown to reduce the incidence of
endocrinopathies like hypogonadism and diabetes.
The benefit of DFO on survival has clearly been
demonstrated by improved survival of subsequent birth
cohorts born after 1960. The age of starting DFO is a key factor
in outcome.
Side Effects
• Local reactions: local reactions like pain, swelling,
itching and erythema are common with DFO infusion.
These are generally due to inadequate dilution or
improper techniques. Ulceration at infusion site can occur
if the needle is inserted intradermally. These reactions can
be managed by varying the injection sites, lowering the
strength of infusion.
• Hearing problems: High doses of DFO have been
associated with bilateral symmetric sensorineural hearing
loss. It is more common in young children with low iron
burden. Milder sensorineural hearing loss is reversible;
hence it is advised to monitor audiometry yearly in patients
on DFO therapy.
• Retinal and optic nerve dysfunction: vision problems
like night blindness, impaired color vision, visual field
defects and reduced visual acuity have been associated
with high doses of DFO. Retinitis pigmentosa may be
seen on fundus examination. Retinal complications are
more common in patients suffering from diabetes or on
concurrent phenothiazine treatment.
• Growth retardation: High doses of DFO in smaller
children can give rise to growth failure. Regular growth
monitoring is thus essential for patients on DFO.
• Skeletal changes: DFO has been associated with
metaphyseal changes particularly in the vertebrae
resulting in short trunk. Radiograph in such patients will
show vertebral demineralization and flatness of vertebral
body. Rickets like bony changes and genu valgum are
other features of DFO induced skeletal changes.
• Others: Rapid intravenous injection can result in flushing.
Renal impairment and interstitial pneumonitis have also
been reported with DFO.
Recommended Therapy
The standard recommended dose of DFO in children is 20–
40 mg/kg/d given as a slow subcutaneous infusion over 8–
12 hours at least 5 days a week.
Dose of DFO may have to be reduced in patients with
low serum ferritin levels. Therapeutic index can be used for
adjusting the doses.
Therapeutic index = mean daily dose (mg/kg)/serum
ferritin (µg/L).
Therapeutic index should be kept below 0.025 to prevent
toxicities due to DFO.
Deferiprone (DFP, L1, 1,2 dimethyl, 3 hydroxy, pyrid-4-one,
Kelfer).
DFP is the first oral iron chelator launched in 1980’s. It was
first licensed for use in thalassemia in India. It is bidentate
iron chelators and binds iron in 3:1 ratio. DFP has a half-life of
1.5 hrs and it is rapidly cleared from plasma by glucuronidation
of iron binding sites in liver. Iron excretion occurs exclusively
in urine (Table 5). Due to its small molecular size and absence
of any net charge on its iron chelate, DFP penetrates the cells
and is able to chelate iron from intracellular compartments
such as lysosomes and mitochondria.
Effect on Serum Ferritin
Pooled analysis of prospective randomized controlled trials
has shown a statistically significant fall in serum ferritin
with DFO in 6 months but no difference was found among
two drugs at 1 year. The effect on serum ferritin is higher at
baseline values of > 2500 µg/L.
Effect on Liver Iron
Various studies have shown considerable variation in effect
of DFP on LIC. This could be due to differences in dosing
schedules, baseline LIC, transfusional iron loading rates and
duration of follow-up.
Effect on Cardiac Iron
Prospective randomized studies have shown that DFP reduces
all degrees of cardiac siderosis. Cardiac T2* improved from
13.3 to 16.5 ms in DFP group compared to 13.3 to 14.4 ms in
DFO group at the end of one year.
In another multicentric study across Italy, there were no
new cardiac events or death due to cardiac cause among
157 patients being treated with DFP alone, compared to 10
cardiac deaths and 42 non-fatal cardiac events occurred in
DFO group.
Adverse Effects
• Arthropathy: Arthropathy due to DFP mainly affects the
large joints, typically knees. Symptoms vary from mild
pain controlled by NSAID’s to severe progressive erosive
arthropathy. Treatment with DFP should be stopped if
symptoms continue to persist despite dose reduction or
are unresponsive to NSAID’s.
• Agranulocytosis: Agranulocytosis (absolute neutrophil
count < 500/mm3 on two consecutive blood samples) is
the most serious adverse effect of DFP. It is observed in 1%
of cases. It is mostly seen in first year of start of treatment
but cases have been reported after 9 years of therapy. The
 Chapter 41  Iron Overload in Chronically Transfused Patients: Diagnosis and Treatment  151
mean duration of neutropenia is approximately 9 days.
Colony stimulating factors can be used to shorten the
duration of neutropenia. It may occur in association with
thrombocytopenia but isolated thrombocytopenia has
also been reported. Agranulocytosis is more common in
patients with Diamond Blackfan anemia. Rechallenge with
the drug should not be attempted. The recommendation
is to check weekly blood counts for all patients on DFP
during the first year of start of treatment and every 2 weeks
thereafter.
  Lesser degrees of neutropenia (ANC between 500 and
1500/mm3) is more common and re-challenge can be tried.
• Gastrointestinal adverse effects: nausea, vomiting and
abdominal pain are the most common adverse effects
seen in patients on DFP. Transient elevation in serum
transaminases can be seen in up to 7% of patients which
settles on withdrawing the drug.
• Zinc deficiency: zinc deficiency has been observed in
patients on DFP particularly those with concomitant
diabetes, due to enhanced urinary excretion.
• Neurological effects: Rare adverse effects like ataxia,
nystagmus, and dystonia impaired cognitive effects
and psychomotor retardation has been observed with
overdosing. These effects are reversible on discontinuation
of drug.
• Teratogenicity: DFP should not be used in any sexually
active male or female unless contraception is being used
as it has been shown to be teratogenic in animals. DFP is
contraindicated in pregnancy as well.
Recommended dose: The daily recommended dose of
DFP is 75–100 mg/kg/d in 3–4 divided doses.
Deferasirox
Deferasirox (DFX) is a bis-hydroxyphenyl-triazole. It is a
tridentate iron chelator, two molecules of DFX bind one
iron atom. DFX has along plasma half-life of 9-11 hours,
thus providing a 24 hour chelation cover (Table 5). DFX is
metabolized in liver via glucuronidation. The iron excretion
with DFX is almost exclusively fecal.
Effect on Serum Ferritin
Several prospective randomized studies have shown a dose
dependent effect on serum ferritin. Serum ferritin is stabilized
in patients receiving 20 mg/kg/d whereas at a dose of 30 mg/
kg/d, there is a steady decline in serum ferritin values over 1
year.
Effect on Liver Iron
A dose-dependent effect of DFX was found on LIC. The mean
LIC remained constant over a year at a dose of 20 mg/kg/d.
LIC showed a fall of 8.9 mg/g dry wt over a year at a dose of
30 mg/kg/d. Blood transfusion rates have also been shown to
affect the response to treatment.
Effect on Cardiac Iron
A prospective randomized multicentric study among 194
patients with thalassemia major, has shown improvement
in cardiac T2* in patients with cardiac siderosis (cardiac T2*
between 5 and 20 ms). The myocardial T2* improved from
11.2 ms to 12.9 ms over a period of 1 year. The left ventricular
ejection fraction was normal at baseline and did not show any
change.
Compliance
DFX has been shown to be associated with greater patient
adherence and satisfaction as compared to DFO due to its
once daily dosing and ease of administration.
Adverse Effects
• Skin rash: A generalized maculopapular pruritic rash has
been observed in 11% patients on DFX. Rash is generally
observed within two weeks of initiating treatment.
Mild rash resolves spontaneously, few patients require
temporary discontinuation of drug.
• Gastrointestinal effects: Transient gastrointestinal
disturbances like nausea, vomiting, abdominal pain is
observed in up to 15% patients. The mean duration is
approximately a week.
• Increase in serum creatinine: A transient elevation in
serum creatinine up to 30% above the baseline has been
seen in up to 38% patients on DFX. Serum creatinine
should be checked at least twice before initiating DFX and
monthly thereafter for patients on DFX. These changes are
mostly transient and serum creatinine stabilizes within
twice the upper limit of normal for a few patients. No
progressive renal dysfunction has been seen in patients on
DFX.
• Effects on liver: Mild transient elevation in serum
transaminases can be seen with DFX. DFX is
contraindicated in patients with renal and hepatic
dysfunction. DFX induced hepatic or renal impairment
appears to be more common in older patients with high
risk MDS who may have pre-existing renal or hepatic
dysfunction.
• Other effects: Sensorineural deafness, hyperacusis,
lenticular opacities have been observed in few patients
on DFX. Annual auditory and ophthalmic checkup should
be done in patients on DFX. Safety in pregnancy not
established.
Recommended Dose
DFX can be started at a dose of 20 mg/kg/d after 10–20
transfusions. In patients with pre-existing iron overload, a
152  Section 1  Clinical Hemotherapy
dose of 30 mg/kg/d is recommended. It has to be taken as a
suspension in water or apple juice, once a day before a meal.
Combination Therapy
A number of patients continue to have high iron burden inspite
of regular intake of an iron chelator. Combination of two iron
chelators remains an alternative for such patients. Two iron
chelators can be used at the same time (simultaneously) or
can be used sequentially.
DFO and DFP
The shuttle hypothesis formed the basis of combination
therapy. DFP being a smaller molecule can remove
intracellular iron and pass it over to DFO, to be removed in
urine or feces. DFP can then re-enter cells to chelate more
iron.
Combination of DFO and DFP has been demonstrated to
improve the LVEF and cardiac T2* in patients with cardiac
dysfunction.
DFO and DFX
Both DFX and DFP target the liver iron pool and their
combined treatment may not be additive. There have been
small studies which have shown a decline in serum ferritin
along with improvement in LIC without any additional
adverse effects, but the combination is currently not
recommended for routine use.
DFX and DFP
Combination of two oral iron chelators has been used in few
small studies. It has been shown to result in a fall in serum
ferritin, with improvement in cardiac and liver T2* along
with improvement in LVEF, glucose metabolism and gonadal
dysfunction. Adverse effects observed are similar to the ones
seen with monotherapy.
Iron Chelation in Non Transfusion Dependent
Thalassemias (NTDT)
Iron chelation should be initiated in patients of NTDT ≥10
years of age if LIC > 5 mg/g of dry wt or serum ferritin >
800 µg/L. Deferasirox at 10 mg/kg/ d is the recommended
treatment.
Iron Chelation in Sickle Cell Disease and
Congenital Sideroblastic Anemias
The indications of iron chelation in this group of patients are
similar to thalassemia major. DFO is the first drug of choice
but compliance is usually poor. DFX has been shown to be
safe and effective in SCD.
Iron Chelation in Inherited or
Acquired Aplastic Anemia
DFO is recommended as the drug of choice for patients
with aplastic anemia requiring repeated blood transfusions.
Bleeding and infection at infusion site are common problems.
DFX has been shown to be effective in this group of patients.
An elevation in serum creatinine has been observed especially
in patients on cyclosporine.
Iron Chelation in MDS, Chronic Myelofibrosis, PNH
Iron chelation should be initiated in low or intermediate
risk MDS patients who have received > 20–25 transfusions
or if serum ferritin > 1000 µg/L. DFO and DFX are licensed
for use but DFO is difficult to use due to accompanying
thrombocytopenia in most of these patients and increased
risk of infections at the infusion sites. DFX has to used with
caution as there is risk of hepatic and renal impairment in this
group of patients.
Suggested reading
1. Aydinok Y, Kattamis A, Viprakasit V. Current approach to
iron chelation in children. British Journal of Haematology.
2014;165:745-55.
2. Borgna-Pignatti C, Rugolotto S, De Stefano P, et al. Survival and
complications in patients with thalassemia major treated with
transfusion and deferoxamine. Haematologica. 2004;89:1187-
93.
3. Cappellini MD, Cohen A, Eleftheriou A, Piga A, Porter J, Taher
A. Guidelines for the Clinical Management of Thalassaemia.
2nd Edition (2008). Cyprus.
4. Hoffbrand AV, Taher A, Cappellini MD. How I treat transfusional
iron overload. Blood. 2012;120(18):3657-69.
5. Marsella M, Borgna-Pignatti C. Transfusional iron overload
and iron chelation therapy in thalassemia major and sickle cell
disease. Hematol Oncol Clin N Am 2014;28:703-27.
6. Wood JC. Impact of iron assessment by MRI. Hematology Am
Soc Hematol Educ Program 2011. pp. 443-50.
Transfusion in Sickle Cell
Anemia
Yazdi Italia
Sickle cell anemia is a hereditary hemoglobin disorder found
amongst different tribal populations of India. So far it is a
neglected rural health issue and there is no national policy for
comprehensive sickle cell program. Gujarat is the first state
to include Comprehensive Sickle Cell Program in the state
health services.
When we talk about anemia, we always think about iron
deficiency and treatment starts with iron therapy. But in case of
sickle cell anemia iron is mostly normal unless malnourished.
Hence all the patients, whose blood is not examined for sickle
cell tests are misdiagnosed and mistreated.
Hemoglobin S can cause RBCs to change to a sickle shape.
The sickle cells are rigid. Rigidity and abnormal shape reduce
their ability to be propelled through tiny/narrow capillaries
and formation of entangled masses of cells in larger blood
vessels. The obstruction of the blood flow produces temporary
or permanent organ dysfunction or structural changes.
The sign and symptoms of sickle cell disease is pallor,
frequent jaundice, bone and body ache, enlarged spleen,
retarded growth, frequent infections and dactilytis.
Life span of RBC in sickle cell disease is less than 30 days
instead of 90 to 120 days. Anemia results from the bone
marrow’s inability to produce enough blood cells to keep pace
with the rate of destruction. This is one of the major factors for
mild to moderate anemia in sickle cell disease patients.
Homozygous sickle cell (SS) disease is associated with
a lowered hemoglobin level which is characteristic of each
patient in the clinical steady state. This clinical steady state is a
state at particular hemoglobin level the sickle disease patient
can perform his normal duties, without much discomfort.
This level varies widely between patients and is influenced
by genetic and environmental factors. In disease of African
origin, levels are typically 6–9 g/dl but steady state values in
individuals may vary as widely as 5–11 g/dL.1
In the sickle cell disease associated with the Asian
haplotype, occurring in the eastern Province of Saudi Arabia
and western and central India, steady state hemoglobin levels
are typically 8–11 g/dl,2,3 probably influenced by the higher
42
frequencies of alpha thalassemia and of high levels of fetal
hemoglobin (HbF) in those populations.
These steady state levels are maintained despite rapid
hemolysis by expansion of the bone marrow and greater
erythropoietic turnover, manifest in the peripheral blood by
an increased reticulocytes count. Hemoglobin levels fall if
this erythropoietic response is limited by nutritional factors
(iron, folate or other deficiencies), metabolic abnormalities
(renal impairment), or infections (septicemia, parvovirus-
induced aplastic crisis), with accelerated hemolysis
(chronic hypersplenism) or red cell pooling (acute splenic
sequestration).
The rate of decline is determined by the specific complica­
tion and is most rapid in acute splenic sequestration (falls
of 3-5 g/dl within hours), the aplastic crisis (1 g/dL/day),
or septicemias, and a gradual decline to a new hematologic
equilibrium may occur in chronic hypersplenism, nutritional
deficiencies and chronic renal failure. The symptoms
are determined to some extent by the speed of decline in
hemoglobin levels and in chronic nutritional deficiency or
renal failure, levels as low as 2-4 g/dL may be surprisingly well
tolerated.
The role of transfusion in the management of lowered
hemoglobin levels in SS disease is determined by the
underlying cause and there are often other more appropriate
therapies to address the diagnosis.
Sickle hemoglobin within the red cell behaves with a low
oxygen affinity4,5 releasing more oxygen in the periphery than
HbA. Oxygen affinity of HbS varies widely between patients
with SS disease and is inversely related to hemoglobin level,6
patients with the greatest shift in their oxygen dissociation
curves tending to run lower hemoglobins, not necessarily
because their hemolysis outstrips the bone marrow response
but because at steady state levels, oxygen delivery may be
near normal even at low hemoglobin level.
Because of this variation, hemoglobin levels cannot be
assumed to reflect oxygen delivery and patients should be
treated according to symptoms rather than their hemoglobin
154  Section 1  Clinical Hemotherapy
level. Transfusions requested in patients with hemoglobin
levels ≥ 8.0 g/dl, is difficult to justify outside the context of a
regular chronic transfusion program. Sickle disease patients
should not be treated like thalassemic patients and regular
transfusion is not indicated depending upon the hemoglobin
content.
Blood transfusions can be life saving for sickle cell disease
patients in certain life threatening complication like
• hemolytic crisis
• sequestration crisis
• Aplastic anemia
• Acute chest syndrome
• Stroke etc.
Further blood transfusion also has an important role in
prevention of life threatening conditions in sickle cell disease
patients like stroke, postoperative acute syndrome, etc.
Transfusion is not a harmless procedure but carries risks
of red cell alloimmunization, transfusion acquired infections,
and delayed transfusion reactions and in those receiving
repeated transfusions, there are additional problems of iron
overload and of maintaining venous access. Furthermore, the
unit cost of processing and matching blood and the delivery
of transfusion have to be considered. Even the patients of
sickle cell disease in India are getting free blood as per the
February 2014 directives of NBTC, a judicious use of blood
is recommended. All transfusions should always be weighed
against the risk and benefits to the patients.
It is currently impossible to state whether all transfusions
are clearly indicated and result in patient benefit. Alternative
therapies such as splenectomy for chronic hypersplenism
have been shown to be effective and require assessment in
India where this complication may be more common. There
is an urgent need to document the patients’ steady state
hematological indices, to record reticulocyte counts and
red cell indices and carefully assess the possible causes of
lowered hemoglobin levels. Until such data are available, a
more conservative transfusion policy may benefit both the
patients and the health care delivery services.
Sickle cell disease is one of the major public health
problems in rural India where blood transfusion services are
still under development, where there are maximum numbers
of sickle disease patients. Further, in India we have highest
numbers of patients of sickle cell disease in the world. Thus
there is an urgent need to strengthen blood bank services and
also to avoid unnecessary blood transfusions to sickle cell
disease patients in rural part of India.
Optimal therapy in sickle cell disease requires regular
review of patients when clinically well to document the
steady state levels and provide the baselines against which
to evaluate changes in hematological indices.6 Hence blood
transfusion in sickle cell anemia should not depend upon the
hemoglobin percentage but look at the patient’s symptoms
and vital signs and decide for transfusion, i.e. Treat the
Patient and Not the Number...........
References
1. Hayes RJ, Beckford M, Grandison Y, Mason K, Serjeant BE,
Serjeant GR. The haematology of steady state homozygous
sickle cell disease. Frequency distributions, variation with age
and sex, longitudinal observations. Br J Haematol. 1985;59:369-
82.
2. Padmos MA, Roberts GT, Sackey K, Kulozik A, Bail S, Morris JS,
Serjeant BE, Serjeant GR. Two different forms of homozygous
sickle cell disease occur in Saudi Arabia. Br J Haematol.
1991;79:93-8.
3. Kar BC, Satapathy RK, Kulozik AE, Kulozik M, Sirr S, Serjeant
BE, Serjeant GR. Sickle cell disease in Orissa State, India.
Lancet ii: 1986. pp. 198-201.
4. Seakins M, Gibbs WN, Milner PF, Bertles JF. Erythrocyte Hb-S
concentration. An important factor in the low oxygen affinity of
blood in sickle cell anemia. J. Clin Invest. 1973;52:422-32.
5. Abdu A, Gomez-Marquez, Aldrich TK. The oxygen affinity of
sickle hemoglobin. Respir Physiol Neurobiol. 2008;161(1):92-4.
6. Mehta V, Mistry A, Raicha B, Italia Y, Serjeant G. Transfusion
in Sickle Cell Disease: Experience from a Gujarat Centre. The
Indian Journal of Pediatrics 2013.
Advanced Therapies for
Treatment of Hemophilia
Tarangini D, Anupam Sachdeva
Introduction
Hemophilia is an X-linked congenital coagulation disorder
resulting from deficiency of factor VIII (f VIII) and factor
IX (f IX). The prevalence of hemophilia A and B is 1 in 5000
and 1 in 30,000 male live births respectively.1 Hemophilia
is characterized by spontaneous or provoked bleeding into
joints, muscles, gastrointestinal system and rarely central
nervous system resulting in major morbidity and even
mortality if untreated. The therapy is aimed at treating,
preventing bleeding episodes and related complications,
to preserve and/or restore joint functions and importantly
to integrate patients into a normal social life. Treatment of
hemophilia has evolved over years from supportive care
and transfusion of whole blood or FFP in early nineties to
comprehensive care, specific factor concentrates to immune
tolerance, immunomodulation therapy and gene therapy.
Comprehensive care of hemophiliac patients consists of
treatment and prevention of bleeding, long term management
of hemophiliac arthropathy and other complications of
treatment and the psychosocial support and education
required to manage home treatment.
At present, patients with hemophilia are managed with
optimized treatment schedules based on factor replacement
either prophylactically or on demand protocol. Prophylactic
factor replacement is associated with marked improvement
in quality of life on account of prevention of hemophilic
arthropathy. The most distressing adverse effect observed
with factor replacement (recombinant or plasma derived) is
the development of inhibitory antibodies against the factor
proteins. The reported incidence of inhibitor formation in
severe hemophilia A is estimated to be around 30% and
3-13% in mild to moderate disease.2
Advanced Therapies for Hemophilia
Under Trial
Factor Repletion
Includes approaches aimed at prolonging the half life of
factor concentrate. There are techniques like addition
43
of polyethylene glycol polymers (PEG) or poly sialic and
PEG modified liposomes (Peg Lip f VIII)3 and molecule
modification with genetic fusion with albumin and IgG
Fc moiety where in a carrier protein (albumin or Ig G Fc)
with a longer biological half life is fused to f VIII.4,5 These
techniques are still under trial and may help achieve universal
prophylaxis.
Genetic Therapies
Gene Therapy
On account of its monogenic nature, hemophilia has been
extensively researched in the field of gene therapy. A modest
increase in coagulation factor levels is enough to convert a
severe into a moderate phenotype. Goal of the therapy is the
replacement of a defective gene sequence with a corrected
version to eliminate disease for the lifetime. It consists
of transplantation of genetically modified cells that may
produce functional protein.6
Cell Therapy
Consists of transplantation of living cells into an organism
in order to repair tissue or restore a deficient function. Cell
therapy strategy is based on the use of stem cells given their
indefinite capacity to renew themselves and differentiate to
become cells of several specific cell lines. These procedures
have been conducted in the past mainly with adult stem
cells and, more recently with progenitor cells partially
differentiated from induced pluripotent stem cell (iPSC).
In a recent study conducted by Follenzi et al. demonstrated
that transplantation of healthy mice Kupffer cells or healthy
bone marrow derived mesenchymal stromal cells can restore
factor VIII levels in plasma.7
Premature Termination Codon Suppression
Nonsense mutation in factor VIII and IX causes a severe
bleeding phenotype which is known as premature termination
codons (PTC) caused by base pair substitution. Advances
156  Section 1  Clinical Hemotherapy
in research have identified and developed interventions to
promote ribosome read through PTC in codon to translate
and express full length functional protein. Ateluren (PTC124)
is an oral investigational drug that promoates dose dependent
read through of nonsense codon.8,9
Management of Inhibitors
At present, there is ongoing research on novel methods to
modulate the immune response to f VIII by manipulating
antigen presentation. These include oral or nasal administra­
tion of f VIII peptides, infusion of immature dendritic cells
or infusion of apoptotic fibroblastic cells.10 The strategies
that evade immune response to f VIII are preparation of less
immunogenic f VIII proteins. Immunosuppressive therapy
targeted against either B or T cells is another therapeutic
approach. Increasing T regulatory cells using either protein
replacement or gene therapy is one of the successful
immunosuppressive therapy.11
Management of Hemophilic
Arthropathy
Advanced therapies like chondrocyte implantation and cell
therapies using bioreactors, growth factors, mesenchymal
stem cells and genetically modified cells may be used in the
repair of chondral damage in advanced arthropathic disease.
Conclusion
Advanced therapies are still at an early research phase and
much effort and investment will be required before they can
be applied in a generalized way. Cell therapy approaches hold
significant hope for palliative treatment of disabling articular
sequelae of hemophilic arthropathy.
references
1. Mannucci PM, Tuddenham EGD. The hemophiliac—from
royal genes to gene therapy. N Engl J Med. 2001;344:1773-9.
2. Witmer C, Young G. Factor VIII inhibitors in Hemophilia A:
rationale and latest evidence. Ther Adv Hematol. 2013;4(1):59-
72.
3. Di Minno G, Cerbone AM, Coppola A, et al. Longer-acting factor
VIII to overcome limitations in haemophilia management:
the PEGylated liposomes formulation issue. Haemophilia.
2010;16(1):2-6.
4. Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S: Genetic
fusion to albumin improves the pharmacokinetic properties of
factor IX. Thromb Haemost. 2009;102:634-44.
5. Schulte S, Weimer T, Wormsbaeche W, Kronthaler U, Groener
A, Lang W, Liebing U: Prolonged in-vivo half-life of FVIIa by
fusion to albumin. Blood. 2007;110:924.
6. Liras A, Segovia C and Gabán SA. Advanced therapies for
the treatment of hemophilia: future perspectives. Orphanet
Journal of Rare Diseases. 2012;7:97.
7. Follenzi A, Raut S, Merlin S, Sarkar R, Gupta S. Role of bone
marrow transplantation for correcting hemophilia a in mice.
Blood. 2012;119:5532-42.
8. Hirawat S, Welch EM, Elfring GL, et al. Safety, tolerability, and
pharmacokinetics of PTC124, a nonaminoglycoside nonsense
mutation suppressor, following single- and multiple-dose
administration to healthy male and female adult volunteers. J
Clin Pharmacol. 2007;47(4):430-44.
9. Kerem E, Hirawat S, Armoni S, et al. Effectiveness of PTC124
treatment of cystic fibrosis caused by nonsense mutations: a
prospective phase II trial. Lancet. 2008;372(9640):719-27.
10. Ragni M, Wu W, Liang X, et al. Factor VIII-pulsed dendritic cells
reduce anti-factor VIII antibody formation in the hemophilia A
mouse model. Exp Hematol. 2009;37:744-54.
11. Miao C. Immunomodulation for inhibitors in hemophilia A:
the important role of Treg cells. Expert Rev Hematol. 2010;3:
469-83.
 Intervention in Bleeding Patients
Bleeding Disorders:
Lab Diagnosis
Mohanvir Kaur, Dinesh Ahluwalia
General aspects
Tremendous advances have taken place in the investiga­
tions of cases suffering from bleeding disorders. Bleeding
secondary to local pathology is numerically more common
than bleeding diathesis where patients suffer from a defect in
coagulation cascade, platelets, etc. These are divided into two
broad categories:
1. Inherited bleeding disorders.
2. Acquired bleeding disorders.
Within each of these two groups, depending upon the
nature of defect in hemostatic system, further subdivisions
are done.
• Vessel wall defect
• Platelet defect
– Quantitative
– Qualitative.
• Deficiency of coagulation protein
• Enhanced fibrinolysis
• Variable combinations.
There are many medical situations where laboratory
is requested to carry out bleeding profile. These common
conditions where such requests are made below:
• A case where bleeding diathesis is suspected by clinical
examination, e.g. generalized purpura, multiple site
bleeding, hemarthrosis, etc.
• Family members of given case of bleeding disorders.
• Unexplained single site bleeding of recurrent nature like
menorrhagia, epistaxis, etc.
• Medical conditions where coagulations are known to be
altered, like in liver disease, uremia, septicemia, snake
bites, obstetric emergencies, etc.
• Preoperative evaluation, like in cardiac surgery, neuro­
surgery, other major surgical procedures.
• Patients on anti-coagulant or fibrinolytic drugs, e.g.
Heparin, warfarin, urokinase, etc.
• Primary hematological disorders or patients on cancer
chemotherapy.
44
A wide range laboratory tests are available to assess the
hemostasis. Many of them are extremely elaborate, time
consuming, expensive and often unnecessary. Automation
is available in the field of coagulation and number of
instruments like platelet counter, platelet aggregometer and
fibrinometer.
All available tests for bleeding disorders have limitations,
and need detailed clinical history to know the type of
hemostatic failure responsible for bleeding. Random use of
investigations is insensitive for determination of presence
of pre existing bleeding disorder and prediction of bleeding
during surgery. The emphasis should be on detailed clinical
history. History and examination of the patients are of
utmost importance in choosing, ordering and evaluating the
laboratory test. The historical evaluation includes:
• The type of bleeding (petechiae, purpura and mucosal
bleeding suggest platelet disorder, while hemarthrosis,
subcutaneous and intramuscular hematomas suggest
coagulation disorder).
• The nature of bleeding—spontaneous or post traumatic,
duration and amount of blood loss, frequency of bleeding,
patient’s response to post-traumatic events, delivery and
surgery, need of blood transfusion and response to such
transfusion.
• Family history if any, carefully drawn pedigree charts,
consanguinity, age, sex, etc.
• Presence of local or systemic medical or hematological
illness, e.g.—renal stones, fibroid, cirrhosis of liver,
uremia, SLE, leukemia, aplasia, hypersplenism, etc.
In routine screening tests some congenital disorders may
fail to detect. To control serious bleeding, blood products are
administered. Use of various investigations is advised to know
type of defect in hemostatic mechanism and appropriate use
of blood products is done.
Laboratory investigations
• The screening test
• Specified test.
158  Section 1  Clinical Hemotherapy

Screening Tests • Anti-platelet antibodies

These tests are available in all the moderate size hospitals.
These include a battery of tests which are relatively simple,
comprehensive and sensitive enough to rule out a significant
bleeding diathesis (Table 1). The tests which are used for
screening laboratory are as follows:
• Complete hemogram including the smear examination
• Platelet count
• Bleeding time (Ivy’s Method)
• Clotting time (Lee and White method)
• Prothrombin time (Quick’s method)
• Activated partial thromboplastin time (APTT)
• Thrombin time (TT)
• Factor XIII screen
• Clot retraction test
• D-Dimer test.
Specified Tests
After performing the screening tests and looking at the
results in the screening test one or more of specified tests
are selected. These tests are not always necessary and need
not be available in a small or moderate size hospitals, for
these, samples are sent to reference laboratory. Majority of
these tests are expensive, time consuming, need expensive
instrumentation and technical expertization. These tests
include:
• Factor assay
• Platelet aggregation studies
• Tests for inhibitors
• Platelet factor 3 (PF3) assay
• Plasminogen assay and other tests for fibrinolysis
• Bone marrow.
Basic tests for coagulation are done with no specific
diagnosis in mind. These tests are performed in laboratory
with attempts to mimic in vitro processes that normally
occur in vivo. So investigations of coagulation require
caution in their interpretation. Caution starts from the
collection of blood, calibration of equipment like water bath,
refrigerators and freezers, centrifuges, reagents and buffers,
plastic and glasstubes, pipettes, stopwatches and clocks, and
automated coagulation analysers. Misleading results in blood
coagulation can arise due to carelessness in preanalytical
phase.
Preanalytic phase variables start from collection of venous
blood sample, anticoagulation, time of sample collection,
and transport to the laboratory, centrifugation, preparation
of platelet–poor citrated plasma, storage of plasma and
sample thawing. Collection of venous blood sample should
be done by trained personal and patient should be relaxed
and in warm surrounding. Certain factors such as excessive
stress, vigorous exercise and faulty techniques can cause
significant changes in the interpretation of the test due
to increase in the factor VIII, von Willebrand factors and
fibrinolysis. The venepuncture must be clean and tourniquet
with a light pressure should be applied for a period less than
1 minute. A wide bore needle 21G should be used. Good
quality plastic syringes are preferred to minimize the effect of
contact activation. Blood is thoroughly mixed with 32 g/liter
(0.109 molar trisodium citrate in a ratio of 1:9). For the
estimation of labile factors V and VIII utmost care is required
that samples once taken should be placed on ice and
delivered immediately to laboratory. Platelet poor citrated

Table 1: Showing abnormal results on screening and their interpretation

Platelets
Bleeding
Count WBCT PT APTT TT Presumptive diagnosis Ancillary tests required
Time
Morphology
NN N uA N A N Hemophilia A or Christmas Factor VIII and IX Assay
disease, very rarely factor XII or XI
deficiency
NN A vA N A N von Willebrand’s disease Factor VII, assay. VWF assay and ristocentin
induced platelet aggregation test
NN N vA A A N Deficiency of factors II, V and X Specific assays for the factor, RVV time
NN Ua A A A A Afibrinogenemia Fibrinogen assay Reptilase time
Hypofibrinogenemia or
dysfibrinogenemia
NN N N N N N With abnormal FSF screen test, Assay of factor XIII. If possible a
Factor XIII defect thromboelastrogram
NN A N N N N Glanzmann’s Thrombasthenia Platelet aggregation test with ADP. Platelet
factor-3 assay
R Giant A N N N N Bernard Soulier Syndrome Platelet aggregation test with bovine
fibrinogen and restocetin
Abbreviations: N–Normal, A–Abnormal, uA–Usually abnormal, vA–Variably abnormal, R–Reduced.

plasma should be prepared by centrifugation at 2000 gyri for
15 minutes at 4°C.
Prothrombin Time
Prothrombin time assess the extrinsic and common pathway.
Tissue factor, thromboplastin and calcium are added to
platelet poor citrated plasma. Prothrombin measures efficacy
of V, VII and X on the fibrinogen concentration of plasma.
Recently recombinant thromboplastins are used and they
are replacing the thromboplastin from rabbit brain. For the
control of oral anticoagulation, a preparation is calibrated
against International thromboplastin, hence commercially
available thromboplastin will have its ISI determined and
clearly available. With most rabbit thromboplastin, the
normal range of thromboplastin is between 11 and 15 seconds
and for recombinant thromboplastin it is between 10–12
seconds. Each laboratory should establish its own normal
range. For the purpose of oral anticoagulant monitoring,
international normalized ratio (INR) has been widely
adapted. This overcomes the reagent's variability when using
ISI. A sensitive reagent has low ISI, around 1.0.
The results are expressed in ratio to calculate INR
Test PT = Prothrombin ratio (PTR)
Control PT
International normalized ratio (INR)
INR = [PTR]ISI
Prolongation of PT Indicates
• Administration of oral anticoagulant drugs
• Liver disease particularly obstructive
• Vitamin K deficiency
• DIC
• Very rarely undiagnosed Factor VII, X, V of prothrombin
deficiency or defect.
APPT
Also known as Partial Thromboplastin Time with Kaolin
(PTTK).
APTT test is for checking the efficacy of intrinsic and
common pathways of coagulation in recalcified citrated
plasma. APTT detects the deficiency or defect of clotting
factor II, V, VIII, IX, X, XI, XII and fibrinogen. A standardized
phospholipid is provided to allow the test to be performed
on PPP. The test not only depends on the contact factor
and factor VIII and IX but also on reactions with Factor X,
V, Prothrombin and fibrinogen. It is also sensitive to the
presence of circulating anticoagulants and heparin. The
normal range is 30–40 seconds. The actual time depends on
the reagents used and the duration of preincubation period,
these preanalytic variables greatly alter the sensitivity of the
test to minor and moderate deficiencies of contact activation
Chapter 44  Bleeding Disorders: Lab Diagnosis  159
of the system. So each lab should calculate its own normal
range. The common causes of prolonged APTT are:
• DIC
• Liver disease
• Massive transfusion with stored blood
• Administration of heparin or contamination of sample
with heparin
• Circulating anticoagulant
• Deficiency of coagulation factors other than factor VII.
A long APTT with a normal PT indicates possible deficiency
of factor VIII, IX, XI, or XII. When APPT is prolong, 50:50
mixture of normal and test plasma should be tested. If in this
mixed plasma APPT corrects by more than 50% of difference
between the two clotting time tests, a factor deficiency is
indicated. Poor correction suggests an inhibitor, which may
be either one of the clotting factors or of the lupus type.
e.g. APTT control : 30 sec.
APTT Test : 60 sec.
50:50 mix : 40 sec.
This shows good correction, means there is factor deficiency.
If 50:50 mix : 52 sec.
This shows poor correction, it means there is circulating
inhibitor which includes lupus anticoagulant.
APTT is used as a test for screening of lupus anticoagulant,
different conditions and reagents may be employed to
optimize sensitivity. Lupus anticoagulant is present in up
to 2% of plasma samples and not associated with increased
bleeding. In general the APTT is insensitive to the presence
of low molecular weight heparin (LMWH). The test may
be normal or only modestly prolonged in the phase of
therapeutic LMWH levels in the plasma. Those bleeding due
to overdose of LMWH need assay for antifactor Xa activity.
Factor XII deficiency is amongst the most common causes
of unexpected prolongation of APTT and is present in 2%
of healthy subjects. Mixing studies are done to distinguish
between factor deficiency and the effect of an inhibitor.
Thrombin Time
Thrombin is added to plasma and clotting time is measured.
It is prolonged when the fibrinogen level is very low in the
presence of heparin and heparin like substance and in the
presence of inhibitors FDPs and qualitative abnormality
of fibrinogen. TT is effected by concentration and reaction
of fibrinogen and by presence of inhibitory substances, i.e.
fibrinogen, FDP and heparin. The normal range of TT should
be within 2 seconds of control (15-19 seconds). Times of
20 second and over are definitely abnormal.
Interpretation of Results
Prolonged TT is seen in following conditions:
• Hypofibrinogenemia was found in DIC and more rarely in
a congenital defect of deficiency.
160  Section 1  Clinical Hemotherapy
• Raised concentrations of FDP, as encountered in DIC or
liver disease.
• Extreme prolongation of TT is nearly always due to the
presence of heparin which interferes with the thrombin-
fibrinogen reaction. If the presence of heparin is
suspected, a Repitilase time test should be carried out.
Low molecular weight heparin (LMWH) produces only a
slight prolongation at therapeutic levels.
• Dysfibrinogenemia, found either inherited or acquired (in
liver disease) or physiologically in neonates.
• Hypoalbuminemia.
Fibrinogen Assay
Fibrinogen assay is done by different methods like clotting,
physical and nephrometric, immunological but Clauss assay
technique is commonly used. In this diluted plasma is clotted
with strong thrombin solution, normal range is 2–4 g/liter.
The fibrinogen is usually low in inherited dysfibrinogenemia
and is insensitive to heparin levels unless heparin level is very
high, high levels of FDP more than 190 ug/mL may interfere
with assay.
Bleeding Time (Ivy Method)
A standard incision is made on the volar surface of forearm
and the time the incision bleeds is measured. Cessation of
bleeding indicates the formation of hemostatic plugs which
are in turn dependent on an adequate number of platelets and
on the ability of the platelets to adhere to the subendothelium
and to form aggregates. It should be less than 9 min.
Interpretation
Prolonged bleeding may be due to
• Thrombocytopenia
• Disorders of platelet function
• vWF deficiency/defect
• Vascular abnormalities as found in Ehlers-danlos
syndrome
• Severe deficiency of factor V or XI or afibrinogenemia.
Platelet Count
Manual counts are used routinely in under resourced
laboratories by using freshly prepared 1% ammonium oxalate
as diluting fluid and Neubauer chamber is used. Normal
platelet count is 1,50,000 to 4,50,000/cmm. Automated full
blood counters produce platelet counts with precision.
If the only abnormality is a low platelet count, possible
causes must be investigated. The usual approach is to
perform a bone marrow aspirate to exclude marrow failure
and establish whether megakaryocytes are present. If the
number and morphology of megakaryocytes in the marrow
are normal, further investigations are undertaken to establish
the cause of presumed peripheral destruction of platelets.
Heparin and other drugs are common causes in hospital
practice.
Blood Film Examination
Although platelet count must be carried out in all the cases,
examination of the peripheral blood film is a must as this
acts as a double check on the platelet count and at the same
time it gives an idea about the morphological abnormalities
of the platelets and the presence and absence of the platelet
aggregates. Normally the ratio of platelets to the red cells in
the peripheral film is 1:20 (in the lack of anemia).
The field of coagulation testing has undergone some major
technological and conceptual developments.
Light Transmission Aggregometry
Light transmission aggregometry (LTA) which is recognized
as the gold standard for the measurement of platelet function
and is able to detect the effects of antiplatelet therapy, to
determine platelet function, and used this information to
predict coagulopathy and bleeding in patients undergoing
cardiac surgery. They used values of 70–100% light
transmittance to indicate normal platelet function, 50–69%
mild dysfunction, 40–49% moderate dysfunction, and <40%
indicated severe dysfunction. They found that markedly
abnormal LTA (<40% light transmittance) accurately
predicted patients requiring multiple transfusions.
Thromboelastography
The field of coagulation has undergone major technological
and conceptual developments. This is an analytic method that
enabled a more global assessment of coagulation in whole
blood. The advantage of thromboelastography (TEG) is that
it is assessing clot initiation, amplification, propagation and
fibrinolysis. TEG combines evaluation of plasma components
of coagulation with cellular component. TEG can be used as
a replacement for laboratory assays or can be used alongside
traditional assays.
Thromboelastograph Hemostasis Analyzer (TEG)
It measures the clot's physical properties to determine whether
the patient will have normal hemostasis, will have hemorrhage
or will develop an inappropriate clot formation known as
thrombosis. In this blood sample is placed into cuvette which
rotates gently with a cycle time of 6/min. A sensor connected
with a torsion wire, is inserted into sample. Clot formation
generates a physical connection between the inner surface of
the cup and surface of the sensor. The change of elasticity is
detected by a computer. The TEG can also accurately assess low
molecular weight Heparin and Coumadin, and has the ability
to distinguish surgical bleeding from nonsurgical bleeding.

Information Provided by TEG
• The time it takes for the first fibrin strand to be formed, is
similar to activated clotting time, PT, APTT.
• Gives information about the kinetics of the clot formation.
• The strength of clot
• Quantitative and qualitative information about the hypo­
coagulation, normocoagulation and hypercoagulation
disorder in patients.
• Reaction time (hr) measures the intrinsic pathway hence
factor VIII IX XI and XII.
There are various parameters of TEG (r) tracing, which
measures different stages of clot development:
R : This is time period from initiation of test to
the initial fibrin formation.
K : This represents the dynamics of clot formation;
it is the time from beginning of clot formation
until the amplitude of thromboelastogram
reaches 20 mm.
Alpha angle : This provides information about kinetics of
fibrin build up and cross linking.
MA : Maximum amplitude, reflects strength of clot
which is dependent on number and function
of platelets and its interaction with fibrin.
MA60 : This measures the rate of amplitude 60
minutes after MA and represents the stability
of clot.
EPL : This measures degree of lysis 30 minutes after
MA. (Estimated percentage lysis).
Uses and Benefits of TEG
• TEG has been used in cardiac surgeries, major trauma
and hepatic transplantation. An increase in k prompts
the administration of FFP, a decreased MA prompts
administration of platelets and tear drop configuration of
fibrinolysis leads to administration of antifibrinolytics.
• TEG is well suited for use in surgical procedures which
are associated with high volume of blood loss which
include cardiac surgeries, orthopediatric procedures,
liver transplantation, major vascular surgeries, trauma
surgeries, obstetrics, and pediatric surgery.
• TEG testing reduces the need of inappropriate
transfusions.
• TEG enables rapid diagnosis and appropriate treatment
for bleeding, hence reducing complications and stay in
hospital.
ROTEM
Rotation thromboelastometry is based on rotation of TEG,
which is related, but in some aspects different from classical
TEG. Many limitations of TEG are overcome by the innovative
rotation thromboelastometry (ROTEM). In this the sensor is
Chapter 44  Bleeding Disorders: Lab Diagnosis  161
fixed on the top of rotating shaft, which is guided by a high
precision ball bearing system. In this the cycle time is 10 per
minute. The exact position of axis is detected by reflection of
light by a small mirror, which is attached to the shaft. The loss
of elasticity upon clotting of the sample leads to a change in the
rotation of the shaft. The opto-mechanical detection method
provides good protection against the impact of vibrations
and mechanical shocks. Transportation and installation of
instrument is simple.
Platelet Mapping
Good correlation between Thrombelastography (TEG)
Platelet Mapping and LTA is seen. Perioperative TEG Platelet
Mapping is used as routine service for patients presenting
for surgery who have failed to omit their antiplatelet
medication. Conservative value of <30% platelet inhibition
is considered safe to proceed for surgery. Postponement of
surgery was avoided in 85% patients with recent ingestion
of antiplatelet drugs. In addition, TEG platelet mapping
enables to administer informed rather than empirical platelet
transfusions in both elective and emergency patients. Recent
use of antiplatelet drugs by patients before operation poses a
dilemma as to whether to proceed with surgery or not. TEG
allows rapid quantification of an individual's response to
antiplatelet agents, TEG platelet mapping to be a very useful
perioperative tool, which has led to improved patient care,
empirical platelet transfusions and financial savings in terms
of reduced surgical cancellation.
Other New Technologies
The functional platelet function monitor gives an idea about
the percentage of platelets, which are normal and percentage
of platelets which have a qualitative effect. Sonoclot is another
technique to determine the clotting disorders.
Summary
In the diagnosis of possible bleeding disorders and in the
assessment of perioperative bleeding, reliance should be
placed on laboratory tests alone. All the screening tests of
hemostasis and blood coagulation have limitations, and
interpretation can be complex.
Suggested reading
1. Douglas A. Triplett Coagulation and Bleeding Disorders:
Review and Update Clinical Chemistry. 2000;46:1260-9.
2. Gregory Romney, Michael Glick. An Updated Concept of
Coagulation with Clinical Implications J am Dent Assoc.
2009;140;567-74.
Management of Coagulopathy
in Liver Diseases

Meenu Bajpai
45
The liver is the primary site for synthesis of most procoagulant Defects in Secondary Hemostasis
and anticoagulant factors. When the liver is compromised
there is a decreased synthesis of both categories of the factors Liver is site of:
leading to a tenuous rebalance of hemostasis. These patients • Synthesis of coagulation/anticoagulation factors
have a much lower reserve and are therefore at risk of both • Metabolism of coagulation/anticoagulation factors
hemorrhage and thrombosis. Routine laboratory assays • Gamma decarboxylation of coagulation factors.
are not able to assess the risk of bleeding and therefore it is Liver disease patients may have hypocoagulability or
difficult to plan prophylactic blood component therapy. hypercoagulability.
Chronic liver disease affects primary and secondary Hypocoagulability may be due to vitamin K deficiency
hemostasis as well as the fibrinolytic system. as decreased bile production causes decreased vitamin K
absorption leading to decreased decarboxylation of Factors
II, VII, IX, X. There is an inadequate production of other
Defects in Primary Hemostasis
coagulation factors synthesized in liver along with an
There are changes in number as well as function of platelets
in patients with liver diseases. Thrombocytopenia is due to
increased platelet sequestration by the liver and spleen. There Table 1: Site of synthesis of the coagulation factors
is also a reduced production of thrombopoietin by the liver. Factor name Roman numeral Source
The half life of platelets is also reduced due to auto-antibodies
Fibrinogen I Liver
(more common in patients with HCV infection and primary
biliary cirrhosis). Prothrombin II Liver**
In addition to low platelet counts there are also defects in Tissue factor III Damaged cells
platelet function in these patients. The causes of thrombo­ Calcium IV Gut and bone
cytopathy are as follows.
Preaccelerin V Liver and platelets
Proconvertin VII Liver**
Intrinsic Factors
Antihemophilic VIII Platelets and endothelium
• Defective TxA2 synthesis factor
• Storage pool deficiency Christmas Factor IX Liver**
• Abnormalities of the platelet Gp Ib (proteolysis of receptors
Stuart-Prower X Liver**
by plasmin).
factor
Plasma XI Liver
Extrinsic Factors thromboplastin
• Abnormal HDL antecedent
• Reduced hematocrit Hageman Factor XII Liver
• Endothelium-derived NO and prostacyclin, two potent Fibrin-stabilizing XIII Liver
platelet inhibitors. factor
Hemostasis is maintained in these patients by com­
** Dependent on vitamin K for synthesis in liver
pensatory mechanisms such as increased production of von Source: Nowale TJ, Handford GA (2009). Pathophysiology: Concepts and Appli-
Willebrand factor by the endothelium and diseased liver. cations for Health Care Professional. (3rd edn). McGraw-Hill. NY.
 Chapter 45  Management of Coagulopathy in Liver Diseases  163
Flow chart 1  Secondary hemostasis in liver disease
increased consumption of by ongoing fibrinolysis (­D dimers)
due to increased tPA levels along with low levels of plasmin
inhibitor factor. Liver disease also leads to dysfibrinogenemia
due to increased sialic acid residues on fibrinogen leading to
impaired fibrin polymerization (Flow chart 1).
Hypercoagulability is due to decreased levels of anti-
coagulant factors—Protein C, Protein S, ATIII, protein Z due
Flow chart 2  Fibrinolysis
From ref. Tripodi A, Mannucci PM. J Hepatol. 2007;46:727-33.
to decreased hepatocyte function associated with increased
consumption due to DIC or hyperfibrinolysis. The levels of
coagulation factors VIII and vWF are increased as they are
acute phase reactants and are synthesized by both liver and
endothelial cells. Fibrinogen is also an acute phase reactant
and may be normal or Increased. These alterations lead to a
concomitant thrombophilic predisposition (Flow chart 1).
Fibrinolysis
All factors involved in fibrinloysis are produced in the liver
exceptions being tissue plasminogen activator (tPA) and
plasminogen activator inhibitor-1 (PAI-1). Hyperfibrinolysis
is the result of of alterations in the levels of the various factors
involved. In acute liver failure there may be hypofibrinolysis
dus to elevated PAI-1 (Flow chart 2).
Conclusion
The typical liver disease patient has multiple and opposing
factors that influence hemostasis and clot formation.
This explains the apparent paradox of the prolonged global
coagulation tests and their poor prediction of bleeding risk.
Overview of Management
The management of coagulopathy in liver disease patients
involves the following:
• Red blood cell transfusions
• Vitamin K
164  Section 1  Clinical Hemotherapy
• Fresh frozen plasma
• Platelets
• Cryoprecipitate
• Prothrombin complex concentrate
• Desmopressin
• Aprotonin, tranexamic acid, epsilon amino caproic acid
• Recombinant factor VII
• Topical agents—cyanoacrylates, fibrin glue, and thrombin
• Reduction of portal pressure—Phlebotomy, diuresis
• Surgical techniques.
Red Blood Cell Transfusions
Red blood cell transfusion should be kept to a minimum
with an aim to raise hemoglobin by not more than 8–9 g/dL.
Liver disease patients are often multitransfused and at risk of
alloimmunization. pathy in liver disease patients to control bleeding and manage
Fresh Frozen Plasma
Fresh frozen plasma (FFP) is used to replace procoagulant and
anticoagulant factors and may be given in bleeding patients
in a dose of 10–15 mL/kg body weight every 6–12 hours
depending on response. A level of 30% of factors is required
for coagulation cascade to function; therefore FFP should be
transfused in bolus doses, rather than divided doses.
Issues Related to the Use of Cirrhotic Patients
These patients have an expanded volume and a precarious
fluid balance and are at risk of transfusion associated cardiac
overload (TACO). TACO may be prevented by carrying out
plasma exchange using FFP as the replacement fluid thereby
replacing depleted plasma from the patient with coagulation
factor rich plasma from normal donors.
Another option is the use of prothrombin complex con-
centrates which are intermediate purity pooled plasma
products containing a mixture of vitamin K dependent pro-
teins prepared from cryosupernatant (CPP) by ion exchange
chromatography and contain clotting factors concentrated to
25 times that of normal plasma.
Cryoprecipitate
Cryoprecipitate is used in liver disease patients to replace
depleted fibrinogen. Cryoprecipitate is used in hypofibrino­
genemia to replace fibrinogen when levels fall below
100 µgm/dL. One unit of cryoprecipitate contains 150–250 mg
of fibrinogen. The average dose is 1–2 units per 10 kg body
weight.
Platelets
Platelets are used in liver disease patients with thrombo­
cytopenia as well as patients with thrombocytopathy. The
broad guidelines are given in Table 2.
Table 2: Indications for platelet transfusion
Indication Trigger
Prophylactic Stable patient with no risk factors for 10 × 109/L
bleeding
Risk factor for bleeding is present- 20 × 109/L
pyrexia>38°C, coagulation abnormality,
recent surgery, ventilation, multiple
vascular catheters
Laparotomy, surgery, liver biopsy 50 × 109/L
Therapeutic Active bleeding 50 × 109/L
Pharmacologic Agents
Pharmacologic agents may also be used to manage coagulo­
hyperfibrinolysis.
• Antifibrinolytic Agents
1. Aprotonin—Inhibits serine proteases- plasmin
2. Lysine analogs-Epsilon amino caproic acid
Tranexamic acid (inhibits plasminogen).
• Desmopressin: Releases endogenous FVIIa, vWF, TPA
• Recombinant Factor VIIa
• Vitamin K.
Lysine Analogs-
Epsilon Amino Caproic Acid (EACA)
Tranexamic acid (AMCA)
• Delay fibrinolysis by reducing the binding of plasminogen
to fibrin.
• Normally plasminogen and plasmin bind to fibrin through
lysine binding sites.
• Delay dissolution of the clot.
• Effective on topical administration.
Risks/Complications
• Gastrointestinal complications—nausea, cramping,
diarrhea.
• Potential to cause widespread thrombosis (especially in
setting of DIC).
Aprotonin: Serine Protease Inhibitor
• Bovine lung-derived 58 residue polypeptide with broad
antiprotease activity
• Directed towards trypsin, Kallikrein, thrombin and plasmin
• Activity: Expressed in Kallikrein inactivator units
• Has potent antifibrinolytic activity
• Useful in patients in with hypofibrinogenemia.
Recombinant Factor VIIa
• Is a 2 chain procoagulant of approximately 50,000 mw
• It becomes active when complexed with tissue factor in the
extrinsic clotting cascade
 Chapter 45  Management of Coagulopathy in Liver Diseases  165

• Half life of 2–3 hours
• Dosage ranges from 30 to 120 µgm/Kg
• Dramatic improvement in PT and arrests bleeding
• Effect short-term, very expensive
• May be used prior to procedure/not for prophylaxis.
Vitamin K
• Bile salts and products of pancreatic lipolysis are res­
ponsible for absorbtion of Fat soluble vitamin K.
• It is a cofactor for the post-translational gamma car­
boxylation of coagulation factors II,VII, IX and X as well as
protein C and S.
• Dose in liver disease patients 5 mg oral or 10 mg subcuta-
neous daily over 5 consecutive days.
• Intramuscular route avoided due to risk of hematoma.
• Intravenous route has a higher risk of reactions.
Summary
Hemostasis in liver disease patients is in a rebalanced state and
laboratory values may not correctly represent the coagulation
profile of the patient. Blood component support is imperative
in bleeding patients. In patients who are not actively bleeding
unnecessary transfusions should be avoided. A summary of
guidelines for prophylactic component transfusions in liver
disease patients undergoing invasive procedures is given in
Table 3.

Table 3: Prophylactic management of patients with liver disease Suggested Reading

Prophylaxis before procedures
Platelet count <50 × 109/L
Fibrinogen <100 mg/dL
Coagulation factor deficiency
Prolonged PT and aPTT
Prolonged PT, normal aPTT
Prolonged thrombin time
Dysfibrinogenemia
Administer platelets to increase
50 × 109/L , –75 × 109/L
Administer cryoprecipitate to
maintain fibrinogen > 100 mg/dL
(7–10 units)
Administer vitamin K, 10 mg daily
subcutaneously for 3 days, and
then FFP if factor VII < 10% or other
factors are > 50%
No treatment beyond vitamin K,
if factor VII > 10%
No treatment if fibrinogen
> 100 mg/dL
1. Caldwell SH. Management of coagulopathy in liver disease.
Gastroenterol Hepatol (N Y). 2014;10(5):330-2.
2. Mackavey CL, Hanks R. Hemostasis, coagulation abnormalities,
and liver disease. Crit Care Nurs Clin North Am. 2013;25(4):
435-46.
3. Restellini S, Kherad O, Jairath V, Martel M, Barkun AN. Red
blood cell transfusion is associated with increased rebleeding
in patients with nonvariceal upper gastrointestinal bleeding.
Aliment Pharmacol Ther. 2013;37(3):316-22.
4. Tripodi A, Mannucci PM. Abnormalities of hemostasis in
chronic liver disease: reappraisal of their clinical significance
and need for clinical and laboratory research. J Hepatol. 2007;
46(4):727-33.
5. Weeder PD, Porte RJ, Lisman T. Hemostasis in liver disease:
implications of new concepts for perioperative management.
Transfus Med Rev. 2014;28(3):107-13.
Recombinant Activated Factor
VII in Clinical Practice

Ravneet Kaur, Kshitija Mittal, Tanvi Sood

46
Recombinant factor VIIa (rFVIIa) is US-FDA licensed for use Indications
in patients of hemophilia with inhibitors against exogenous
Factor VIII or IX. However, it is being increasingly used to Approved Indications
prevent and or stop bleeding that is uncorrectable by other
means. United States Food and Drug Administration
rFVIIa is produced by transfection of human factor VII (FDA) Approved Indications
gene in baby hamster kidney cells cultured in bovine albumin
• Treatment or prevention of bleeding in patients with
and has amino acid sequence identical to that of plasma
hemophilia A or B who have inhibitors to factors VIII or IX,
derived factor VII. There is no risk of transfusion transmitted
respectively.
disease with its use as no human or animal source is involved
• Treatment or prevention of bleeding episodes in patients
in its production.
with acquired hemophilia.
• Treatment or prevention of bleeding episodes in patients
Mechanism of action with congenital factor VII deficiency.

Hemostastis is a physiological mechanism that maintains European Approved Indications

blood in a fluid state within circulation. Initiation of
hemostasis occurs by the exposure of tissue factor (TF) to • Bypassing inhibitors to factors VIII and IX in patients with
vessel wall following an injury. Following this, TF forms hemophilia A and B
complex with FVII and FVIIa on the surface of TF bearing • Treatment of congenital factor VII deficiency
cells. TF-VIIa complex converts Factor X to Xa and Factor IX to • Glanzmann’s thrombasthenia.
IXa. This results in generation of limited amount of thrombin
which activates platelets. Activated platelets further support Off-label uses
the coagulation factors (Factor VIII to VIIIa, Factor V to • Traumatic bleeding
Factor V a) and thereby thrombin burst is generated leading • Perioperative blood loss
to effective hemostasis. • Postpartum hemorrhage
Administration of pharmacological dose of exogenous • Spontaneous intracerebral hemorrhage
rFVIIa bypasses factor VIII or factor IX and directly activates • Bleeding in liver diseases
factor X. As a result there is enhanced thrombin generation • Anticoagulant associated bleeding.
on the activated platelet surface at the site of injury. This
increased rate of thrombin along with large doses of rFVIIa Pharmacokinetics, Dosing and
leads to formation of a tight fibrin hemostatic plug, which
is less susceptible to fibrinolysis. It is postulated that the
Laboratory Monitoring
decrease in fibrinolysis is due to increase in thrombin- rFVIIa has a short half life of 2.5 hours. Activity of rFVIIa
activatable fibrinolysis inhibitor and increase in FXIIIa. decreases in the presence of decreased levels of other
 Chapter 46  Recombinant Activated Factor VII in Clinical Practice  167
coagulation factors, thrombocytopenia and acidosis. Its
activity is maintained at lower temperature when overall
hemostatic function of the body is impaired.
Optimal dose and dosing regimen is still not well defined for
rFVIIa. The standard modality is intermittent infusion of rFVIIa
every 2 to 6 hours. However, studies have reported continuous
infusion to be more effective for maintaining rFVIIa levels.
Laboratory monitoring of hemostatic efficacy in patients
treated with rFVIIa is complex. Standard PT and aPTT test do
not correlate well with the clinical picture. A modified version
of the Staclot VIIa-rTF (Diagnostica Stago, France) assay is
being used for monitoring patients on treatment with rFVIIa.
Thromboelastography has also shown promising results in
monitoring of hemostatic effects when using rFVIIa.
Side Effects
Few side effects in the form of thromboembolic events have
been reported with the use of rFVIIa in hemophilia patients
with inhibitors. However, more complications/events have
been reported with the off label use of rFVIIa.
rFVIIa is being used as a general hemostatic agent to
enhance hemostasis in patients with bleeding who may
or may not have coagulation defects. However, the drug
is expensive. The literature reports no survival benefit in
patients with off label indications. Thus, it should be used
cautiously in non approved situations.
Suggested reading
1. Dutta TK, Verma SP. Rational use of recombinant Factor VIIa
in clinical practice. Indian J Hematol Blood Transfus. 2014;30:
85-90.
2. Midathada MV, Metha P, WanerM, Fink LM. Recombinant
Factor VIIa in the treatment of bleeding. Am J Clin Pathol.
2004;121:124-37.
3. Poon MC. Use of recombinant Factor VIIa in hereditary
bleeding disorders. Curr Opin Hematol. 2001;8:312-8.
Plasma Versus Recombinant
Clotting Factors
Rajesh C Gopal
Management of coagulation disorders, say hemophilia has
come a very long way with the advancement of transfusion
medicine and myriad technological breakthroughs support­
ing its pursuits.
The two nightmarish and relatively common coagulation
disorders viz., hemophilia A and hemophilia B have
been traditionally managed with the ever increasing
armamentarium which has included but is not limited to:
• ‘Fresh’ whole blood
• Fresh frozen plasma
• Low or intermediate potency, cryoprecipitate
• High potency human factor VIII, animal factor VIII
• Recombinant Clotting Factors: the availability of these concentrates led to home treatment,
– Recombinant human factor VIII
– Licensed recombinant factor IX product
– Recombinant activated factor VII.
Till the 1960s, fresh frozen plasma (FFP) was the mainstay
of treatment for hemophilia A and hemophilia B. One bag of
FFP contains only small amounts of factor VIII and factor IX,
thus large volumes of intravenously administered FFP are
required to effectively manage bleeding episodes. Children
generally need to be hospitalized for treatment of bleeding
into a knee, an elbow, or any other joint.
Clotting factor concentrate
Plasma factor concentrate is made from human plasma. The
plasma is processed to separate the clotting factors from the
other parts of the plasma. This creates the clotting factor
concentrate. The concentrate is processed to kill any viruses
that might have been in the plasma.
Demerits of Plasma factor
concentrates
Inhibitors
Some people develop antibodies to the injected clotting
factors. These antibodies are called inhibitors. If one develops
47
inhibitors, the usual forms of clotting factors may not
effectively prevent or stop bleeding. Children are more likely
to develop inhibitors than adults.
Viral Infections/TTIs
By the late 1960s, scientists and manufacturers developed
methods for separating factor VIII and factor IX from pooled
plasma, resulting in neatly packaged bottles of freeze-dried
(lyophilized) factor VIII or factor IX concentrates. Each bottle
had a label indicating the amount of factor VIII or factor IX it
contained, allowing more accurate dosing. By the early 1970s,
greatly changing the lives of people with hemophilia.
However, there was a big price to be paid for this
advancement. Thousands of plasma donations were
combined for preparation of one batch of plasma-derived
factor VIII or factor IX concentrate. In the early 1980s, human
blood, plasma, and plasma-derived products were found
to be the responsible for transmission of potentially lethal
blood-borne viruses, including hepatitis viruses and HIV.
Manufacturers of plasma-derived clotting factor concentrates
did make all efforts to kill these viruses with dry heat, solvent-
detergent treatment and pasteurization, but could not
succeed in inactivating all the pathogens. In the USA, by
mid-eighties most patients with hemophilia were switched
to heat-treated concentrates, but many of them had already
acquired HIV infection and that ultimately proved fatal for a
big percentage of them. Safety of plasma-derived products
in the hemophilia community emerged as a major concern
amongst all the stakeholders.
Subsequently better screening methods for blood donors
were developed, improving the safety of donated plasma.
Screening of donors for the hepatitis B virus was already in
place, and in 1989 the hepatitis C virus (HCV) was isolated,
allowing HCV antibody testing of donors to begin in 1990.
HIV was identified in 1984, and by 1985 a blood test for HIV
antibodies was instituted in blood and plasma collection
facilities.
 Chapter 47  Plasma Versus Recombinant Clotting Factors  169

A great advancement came in the mid-1960s with
the discovery of a method for preparing factor VIII from
FFP by allowing it to thaw in the cold (cryoprecipitated
plasma). This preparation could be stored in frozen form as
“cryoprecipitate.” This allowed intravenous administration
of more factor VIII in a smaller volume, allowing outpatient
treatment for bleeds and even elective surgery in persons
with hemophilia A. This more concentrated form of clotting
factor VIII rapidly became the preferred treatment for acute
bleeding episodes in patients with hemophilia A.
The risk of getting a viral infection from plasma factor
concentrates is very low. These products are processed to kill
viruses. The products are also tested to make sure they do not
contain viruses.
The successful cloning of the factor VIII gene in 1984 was
a major breakthrough, allowing production of recombinant
human factor VIII (r factor VIII). It was followed by clinical
trials in humans.
By 1992, two pharmaceutical companies had licensed r
factor VIII products for use in hemophilia A. Cloning of factor
IX was first reported in 1982, and a licensed r factor IX product
(Bene FIX) became available for people with hemophilia B in
1997.
Other significant advances that have been made in recent
years include treatment for patients with inhibitors (anti­
bodies that inhibit or interfere with the function of factor VIII
or factor IX) and prophylaxis (treatment to prevent disease).
Inhibitor antibodies develop in approximately 30–35% of
people with hemophilia A and 1–3% with hemophilia B.
Scientists have also developed innovative treatment
products for ‘bypassing’ the inhibitor, such as recombinant
activated factor VII (r factor VIIa), which was first licensed for
use in hemophilia in 1997.
Newer second-generation products to treat inhibitors are
actively being developed, and while much progress has been
made in treating  inhibitors, this complication remains the
greatest problem in the management of hemophilia today.
With safer clotting factor replacement products, pre­
ventive (prophylactic) treatment is being widely accepted
thereby resulting in larger preservation of normal joint and
musculoskeletal function in adolescents and young men with
hemophilia.
There is almost no risk of an infection from a recombinant
clotting factor. Recombinant clotting factors are made in a
laboratory/by genetic engineering. They are not obtained
from blood and are made with recombinant DNA technology.
They are concentrated into a powder form that is then mixed
with sterile water and injected.
Since their transition into the clinic, the recombinant
versions of both factor VIII and IX have proven to be
remarkable facsimiles of their plasma-derived counterparts.
The broad adoption of recombinant therapy throughout the
developed world has significantly increased the supply of
clotting factor concentrates and helped advance aggressive
therapeutic interventions such as prophylaxis.
Incidentally, the recombinant era for hemophilia began in
the early 1980s with the cloning and subsequent expression
of functional proteins for both factors VIII and IX. Efficient
production of recombinant clotting factors in mammalian cell
culture systems required overcoming significant challenges
due to the complex post-translational modifications that
were integral to their pro-coagulant function. The quick
development and commercialization of recombinant
clotting factors was, in part, facilitated by the catastrophic
impact of viral contamination of plasma-derived clotting
factor concentrates at the time. By 1992, two pharmaceutical
companies had licensed recombinant factor VIII products for
use in hemophilia A. Cloning of factor IX was first reported
in 1982, and a licensed recombinant factor IX product (Bene
FIX) became available for people with hemophilia B in 1997.
The major on-going challenges in hemophilia care have
been effectively addressed by recombinant DNA technology
as follows:
• Reduced costs of therapy
• Increased availability in the developing world
• Improved functional properties of these proteins.
On June 26, 2013, the US. Food and Drug Administration
approved Rixubis [Coagulation Factor IX (Recombinant)] for
use in people with hemophilia B who are 16 years of age and

S.No. Blood component/FFP Recombinant blood product/clotting factors

1. Manufactured in a blood component separation unit Prepared in a Recombinant Technology/Genetic Engineering Laboratory
2. High cost of preparation of coagulation factor concentrates Cloning of gene for say factor VIII and Recombinant technology have
been major break throughs with reduction of cost of clotting factors
due to mass production
3. Complications of plasma component infusions include pyro- Pyrogenic reactions are uncommon and allergic reactions, though
genic and allergic (especially to plasma proteins) reactions known, are extremely rare with recombinant clotting factors
4. Blood component or concentrates/cryoprecipitate devel- Recombinant clotting factors have practically minimized the likelihood
oped have a higher risk of development of inhibitors due to of inhibitors with a bright future of long-term prophylactic replacement
antibody formation therapy in inherited disorders especially severe hemophilia A and
Christmas disease
5. Many hemophiliac patients develop hepatitis B and AIDS Advent of Recombinant clotting factors has effectively addressed the
over a period of time besides the possibility of transmission issue of transmissibility of infections by infusion/transfusion of clotting
of myriad TTIs due regular component therapy factors
170  Section 1  Clinical Hemotherapy
older. Rixubis is indicated for the control and prevention of
bleeding episodes, perioperative (period extending from the
time of hospitalization for surgery to the time of discharge)
management, and routine use to prevent or reduce the
frequency of bleeding episodes (prophylaxis). Although serious
side effects including anaphylaxis (life-threatening allergic
reactions) can occur, the most common side effects observed
in patients in clinical studies were dysgeusia (distorted taste),
pain in an extremity, and atypical blood test results.
The development of recombinant VIIa has been a further
advance bringing a recombinant option to hemophilia
patients with inhibitors.
Conclusion
Recombinant clotting factors provide a definite edge over
Plasma in both the management of bleeding in people of
hemophilia as well as prevention of deterioration of joint and
musculoskeletal functions.
Suggested Reading
1. http://www.webmd.com/a-to-z-guides/clotting-factor-
replacement-for-hemophilia.
2. h t t p : / / w w w . f d a . g ov / Ne w s Ev e n t s / Ne w s ro o m / P re s s
Announcements/ucm358918.htm.
3. http://www.hematology.org/About/History/50-Years/1524.
aspx
4. http://www.hemophilia-information.com/blood-clotting-
proteins.html
5. htt p : / / w w w . h e m o p h i l i a. ca / e n / b l e e d i ng - d i s o rd e r s /
hemophilia-a-and-b/the-treatment-of-hemophilia/factor-
replacement-therapy/
6. http://www.hemophilia.ca/files/Chapter%2005.pdf
7. http://www.rch.org.au/bloodtrans/about_blood_products/
Clotting_Factor_Concentrates/
8. http://www.bcbsms.com/com/bcbsms/apps/PolicySearch/
views/ViewPolicy.php?&noprint=yes&path=%2Fpolicy%2Fem
ed%2FHemophilia+Factor+VIII+(Human%2C+Recombinant
%2C+Porcine)+and+Factor+IX+(Human%2C+Complex%2C+
Recombinant).html
Solvent Detergent Treated
Plasma in Clinical Use

M Joshua Daniel Jeyakumar, Suryatapa Saha, Prakash H Muddegowda, Prathiba L Pujjita

48
Introduction SD plasma can be stored for up to one year frozen at –18° C.
When ordered for transfusion it is thawed in a  water bath
The main indication for the transfusion of plasma is to to a temperature of 37° C, which takes approximately 25 to
correct deficiencies of clotting factors, for which a specific 30 minutes and can be kept refrigerated for up to 24 hours at
concentrate is not available, in patients with active bleeding. 1° to 6° C.
Viral safety depends on donor selection and screening;
thus, there continues to be a small but defined risk of viral Table 1: Difference between FFP and SD Plasma
transmission comparable with that exhibited by whole blood. Parameter FFP Solvent detergent
Screening for transfusion transmitted infections (TTIs) to plasma (Octaplas)
exclude blood products at risk of transmitting infections from Plasmin inhibitor U/dL 72–132 24 (20–27 )*
donors to recipients is a critical part of the process of blood Antitrypsin activity g/L 0.95–1.30 0.83 (0.73–0.92) **
transfusion service.
The solvent detergent (SD) method to inactivate enveloped Protein S activity U/dL 56–168 64 (55–71) *
viruses in plasma protein preparations was first developed Protein S total 0.92 (0.76–1.18) 0.78 (0.65–1.00) **
in the early 1980s. The method proved effective in the antigen U/mL
processing of coagulation factor concentrates by disrupting C3a des-Arg ng/mL 190–976 1927 (923–2993) **
the membranes of lipid-enveloped viruses, cells and most Lipoprotein (a) mg/dL 0.3–34.1 5.5 (2.8–8.6) **
protozoa, while leaving the labile coagulation factors intact.
Fibrin monomer mg/mL 0–22 34 (29–40) **
Its efficacy with respect to bacteria is more variable generally,
and it is ineffective against non-lipid-enveloped viruses. The Citrate mM 17.5 (14.2–20.9)*
SD process is the most widely used and best validated and *n = 12 **n = 8 ***n = 2
robust pathogen inactivation technology known today, and
many million doses of SD-treated plasma proteins have been Properties of SD Plasma
transfused without any report of transmission of enveloped
viruses but does not inactivate non-enveloped viruses such Solvent detergent-fresh frozen blood plasm (SD-FFP) is a
as Hepatitis A virus (HAV) or Parvovirus. pharmaceutical product, obtained from a pool of about 1,000
units of FFP, with the following characteristics:
Production of SD Plasma • High batch per batch standardization.
• Declared concentration/activity of the biologically active
SD plasma is a blood product that has undergone treatment proteins.
with the solvent tri-N-butyl phosphate (TNBP) and the • Reduced immunological risks related to the presence of
detergent Triton X-100 to destroy any lipid bound viruses antibodies, cells (or their fragments).
including: HIV1 and 2, HCV, HBV and HTLV I and II. The SD • Inactivation of the majority of potentially transmissible
process includes the  pooling of up to 1000 units  of thawed pathogens.
fresh frozen blood plasma (FFP), treating it with the solvent • Selective elimination of units contaminated by hepatitis A
and detergent (Table 1). The treated Blood plasma pool is virus or parvovirus B19.
then sterile filtered (and thus leukocyte-reduced) before • Lower levels of Factor V, Factor VIII, Protein S, Anti-
being repackaged into 200 mL aliquots or bags and refrozen. plasmin and Anti-trypsin.
172  Section 1  Clinical Hemotherapy

Octaplas Follow-Up Study Table 2: Adverse reactions data on use of SD plasma

• 1201 consecutive open heart surgical procedures in 1169 Blood product Number of Mild reactions Serious TRALI
transfusions reactions
patients
– Adults: 921 Erythrocytes 1.552.216 1748 / 114.8* 101 / 6.5* 5
- Transfused 610 (66%) Thrombocytes 157.858 462/292* 20 / 12.7* 4
- Transfused with Octaplas: 450 Octaplas 350.751 65/18.5* 6 / 1.7* 0
– Octaplas only: 108
*n = 12
- Children (≤15 years): 279 Transfused 198 (71%)
- Transfused with Octaplas: 122
– Octaplas only: 63. Risk of Fibrinolysis
The study showed In 2003, de Jonge et al reported laboratory signs of fibrinolysis
• That Octaplas is an adequate substitute for FFP in open during OLT when using Octaplas, but no significant differences
heart surgery in blood loss. After introduction of low dose Aprotinin neither
• Only mild allergic reactions venous thromboembolism (VTE) nor fibrinolysis occurred in
• No red blood cell antibodies 120 subsequent OLT treated with Octaplas.
• Viral safety was specially analyzed in 343 patients with
adequate preoperative and 6–12 months follow-up blood Risk of Venous Thromboembolism
samples
– This analysis demonstrated no transmission of viral • In Norway where more than 3000 PE have been performed
disease caused by: HBV, HCV, HIV, HTLV, CMV or HAV with Octaplas, we consider TTP patients to be at risk
and Parvovirus. for VTE when thrombocytes normalize during PE, and
• Octaplas in liver transplantation therefore routinely take precautions with
– The period 2005-2009 was specially analyzed • LMW heparin
– 250 consecutively transplanted patients (214 adults, 36 • Elastic stockings.
children)
– No major thromboembolic events Pharmacoeconomics
– No episodes of TRALI (or other severe transfusion
Cost-effectiveness of European SD plasma
reactions)
• Because of low residual risk for recognized viral infections,
• Plasma exchange with octaplas
cost-effectiveness of pathogen reduction is low in
– Over 3000 plasma exchanges with Octaplas (10–20
industrialized countries.
patients per year since 1993).
• Cost of a quality adjusted life year is over US $ 7.5 million.
– In 2010-2011: 435 plasma exchanges were performed
– Cost-effectiveness of SD treatment combined with
on TTP/HUS patients, and in no instances was there
elimination of TRALI and significant reduction of
observed venous thromboembolism as a complication
allergic/anaphylactic adverse events (Octaplas/
of the plasma exchange.
Octaplas LG)
Experience: Octaplas is safe and effective, allergic reactions • Depending on the estimated frequency of TRALI the cost
are only rarely observed (Table 2). of a quality adjusted life year has been calculated between
US $ 8315 and US $ 40000.
Clinical experience and studies with European SD plasma
• 18 studies and retrospective analyses (4 prospective
randomized) covering all indications for European SD
Conclusion with European SD plasma
plasma, document the excellent clinical efficacy and • Over 12.5 million bags of Octaplas or licensees have been
tolerance of Octaplas. transfused to more than 4 million patients
• The studies included different kind of patients • Octaplas can be used in all indications for all patient
– Coagulopathy/DIC associated with surgical bleeding, groups including newborns
cardiac surgery, obstetric complications, intrauterine – TTP and liver transplant patients can be transfused
transfusions, liver disease or liver transplantation. safely
– Inherited coagulation disorders • There is no need for regular FFP
– Plasma exchange due to TTP or immune modulation • Special blood cell components including exchange trans-
– Neonates, children and adults. fusions, can be prepared in Octaplas
 Chapter 48  Solvent Detergent Treated Plasma in Clinical Use  173
• Incidence of anaphylactic and allergic reactions including
serious adverse reactions, are significantly reduced
• No TRALI reactions have been documented
• No cases of virus disease transmission have been reported
• Hospital blood banks and clinicians are satisfied with the
safety and clinical efficacy of the standardized product
with a predictable effect
Suggested Reading
1. Edel E, Al-Ali HK. Efficacy and safety profile of solvent/
detergent plasma in the treatment of Acute thrombocytopenic
Purpura. Transfus Med Hemother. 2010;37(1):13-9.
2. Flamholz R, Hye-Ran Jeon, Baron JM. Study of three patients
with Thrombotic Thrombocytopenic Purpura exchanged with
solvent/detergent treated plasma: Is its decreased protein S
activity clinically related to their development of deep venous
thrombosis, Journal of clinical apheresis. 2000;15(3):169-72.
3. Giancarlo Liumbruno, Angela Lattenzio, Gina Rossetti. Recom­
mendations for the transfusion of plasma and platelets, Italian
society of Transfusion Medicine and Immunohaematology.
2009;7(2):132-50.
4. Hellstern P, Solheim BG. Use of Solvent detergent treatment
in pathogen reduction of plasma. Transfus Med Hemother.
2011;38(1):65-70.
5. Lerner RG, Nelson J, Sorcia E, Grima K. Evaluation of solvent/
Detergent treated plasma in patients with a prolonged
prothrombin time, Vox Sanguinis. 2000;15(3):161-7.
6. Riedler GF, Haycox AR, Duggan AK, Dakin HA. Cost
effectiveness of solvent/detergent-treated fresh frozen plasma,
Vox Sanguinis, 2003. p. 85.
7. Sinnott P, Bodger S, Gupta A. Presence of HLA antibodies
in single donor derived FFP compared with pooled solvent
detergent treated plasma (Octaplas), European Journal of
Immunogenetics. 2004;31(6):271-4.
8. Theusinger OM, Baulig W, Seifert B, Spahn DR. Relative
concentrations of haemostatic factors and cytokines in solvent/
detergent treated plasma and FFP, International Scientific
Meeting in Anaesthesiology, 2013.
9. Ulrich JH Sachs, Dorte Kauschat. White blood cell reactive
antibodies are undetectable in solvent/detergent plasma,
Transfusion. 2005;45(10):1628-31.
10. WHO Technical Report: Guidelines on viral inactivation and
removal procedures intended to assure the viral safety of
human blood plasma products, World Health Organization,
Series no-924,2004.
Cryoprecipitate

Aradhna Sharma
49
Cryoprecipitate is the common term for the cold insoluble • Factor XIII deficiency
plasma proteins that precipitate in fresh frozen plasma when • Source of Fibrin Glue used as topical hemostatic agent
it is thawed between 1°C and 6°C and then centrifuged, in surgical procedures and to remove fragmented renal
collected and refrozen. calculi.
Initially developed in the mid-1960s by Judith Pool and • Uremic coagulopathy
colleagues, cryoprecipitate revolutionized the treatment of • Reversal of thrombolytic therapy
hemophilia and von Willebrand disease (vWD), previously • Massive transfusion with bleeding.
dependent on whole blood or fresh plasma.
Common misuses
Cryoprecipitate constituents
• Fibrinogen replacement with normal fibrinogen levels and
and their stability no evidence of increased dysfunction or consumption.
The main constituents of cryoprecipitate are the high • Reversal of Warfarin therapy.
molecular-weight proteins fibrinogen, factor VIII, von- • Treatment of impaired surgical hemostasis in the absence
Willebrand factor (vWF), factor IX, and fibronectin. Remain­ of hypofibrinogenemia.
ing components include small amounts of immunoglobulin • Treatment of hepatic coagulopathy with multiple factor
G, IgM, and albumin. deficiencies.
Each bag has approximately.
Plasma : 10–15 mL Doses and Administration of
Factor VIII : 80–100 IU Factor VIII Concentrates
Fibrinogen : 150–250 mg Cryoprecipitate contains a negligible amount of red blood
von-Willebrand Factor : 40–70% cells and only a small amount of the isohemagglutinins anti
Fibronectin : 55 mgm A/or anti B so cryo may not be ABO identical, however, ABO
compatible cryoprecipitate is preferred. The compatibility
Factor XIII : 20–30% of the original
testing is not necessary, Rh type need not be considered when
Several studies show that fibrinogen levels remain stable using this component.
and adequate up to 24 hrs after thaw at room temperature In neonatal recipients, cryoprecipitate should be ABO
and in units kept up to 8 years in frozen storage.1 In contrast, compatible because of the smaller plasma volumes in infants.
factor VIII levels appear less stable than fibrinogen over time.2 A dose of 1 unit of cryoprecipitate per 10 kg body weight
can be expected to increase the fibrinogen concentration by
Indications approximately 50 mg/dL in the absence of heavy consumption
or bleeding. The frequency and duration of administration is
• Hemophilia A based on the underlying condition.
• von Willebrand disease Factor VIII content of cryoprecipitate varies with each
• Congenital or acquired fibrinogen deficiency donor and blood group. In blood group A, factor VIII level is
• Acquired factor VIII deficiency (e.g. DIC, massive usually found more than other groups.3
transfusion)
 Chapter 49 Cryoprecipitate  175

In calculating the dose, it can be assumed that 1 IU of Factor VIII assay should be done to serve as a guide to
factor VIII/kg body weight will increase the patients factor therapy. In emergencies, the aPTT can be used as a rough
by 2% (0.02 IU/mL). Thus, if the patients base line factor VIII guide to factor VIII activity.
level is 0.01 IU/mL, a dose of 20 IU/kg body weight would be In mild or moderate hemophilia A, Desmopressin
expected to raise the level to 40% (0.4 IU /mL). (DDAVP) 0.3 ug/kg intravenous, is the treatment of choice.
The half-life of factor VIII is 10-12 hours. Antifibrinolytic agents (aminocaproic acid and tranexamic
If 20 units/kg body weight are required, a person of 70 kg acid) are useful in controlling bleeding in oral cavity. The
weight will require 1400 IU of factor VIII. recommended dose of aminocaproic acid is 75 mg/kg body
CRYO could be used to supply 1400 IU of factor VIII, but at weight every 4–6 hours, that of tranexamic acid is 25 mg/kg
80 IU per bag this would require at least 17–18 bags of cryo. body weight every 6–8 hours. Antifibrinolytic agents are given
1 day preoperative and continued for 7–10 day. The therapy of
The amount of factor VIII required can also be calculated as
choice for severe hemophilia A is Factor VIII concentrate. Factor
follows:4
VIII concentrate, 500 IU bottle, available as pharmaceutical
Weight (kg) × 70 mL/kg : Blood volume (mL) product, is the product of choice for most of the hemophiliacs.
Blood volume (mL) × (1.0- : Plasma volume (mL)
Hct) Preparation, storage and
Units of factor VIII required : (Desired factor VIII level in units/mL quality control
Initial level of factor VIII/mL) × Blood is collected in a triple CPD/CPDA blood collection
plasma volume in (mL)
bag and fresh frozen plasma is separated. Frozen plasma
for cryoprecipitate preparation may be stored for up to 12
Example months at −30oC. The cryoprecipitate may be prepared at any
time during these twelve months.
A 70 kg severe hemophiliac with hematocrit of 40% has initial
Frozen plasma is allowed to thaw in a thawing water
factor VIII level of 2 IU per dl (0.02) IU/mL, 2% activity). How
bath at 4oC or in a refrigerator overnight. Overnight thawing
many units of factor VIII concentrate should be given to raise
process results in a plasma protein precipitation rich in factor
his factor VIII level to 50 IU/dl?
VIII, in the form of white flakes of varying size.
70 kg × 70 mL/kg : 4900 mL (Blood volume) The bags are removed and centrifuged at 5000 × for five
minutes at 4oC (heavy spin). The supernatant plasma is
4900 × (1.0-0.40) : 2940 mL (Plasma volume)
separated into transfer bag by hanging the bag in the inverted
2940 × (0.50-0.02) : 1411 IU of Factor VIII required position, leaving the cryoprecipitate adhering to the walls of
bag.
This dose will increase the level to 50 IU/dl. The tubing is sealed and the cryoprecipitate bag is
The duration of treatment with factor VIII depends upon separated immediately. The cryoprecipitate is stored at −18oC,
the type and location of hemorrhage. After major surgery, the transported in a frozen state, and used within 12 months of
factor VIII level should be maintained above 40–50 IU/dl for the original date of collection.
at least 10 days. To assure the quality of cryoprecipitate, the CFR dictates
that quality of antihemophilic factor and fibrinogen should
be tested monthly on at least four representative containers of
Hemophilia A- Recommended Doses of Factor VIII
cryoprecipitate. With every quality assurance testing, the AABB
Type of bleeding Doses Frequency standard states that unpooled components of cryoprecipitate
(Factor antihemophilic factor should contain a minimum of 150 mg of
VIII IU/kg)
fibrinogen and 80 IU of coagulation factor VIII.
Acute hemarthrosis
Early 10 Seldom required Quality control of cryoprecipitate
Late 20 20 IU every 12 hours Parameter Quality control Frequency of control
Intramuscular hemorrhage 20–30 20 IU every 12 hours Volume 10–20 mL Occasionally
Life-threatening situation 50 25-30 IU every
Factor-VIII 80–120 units Occasionally
Intracranial hemorrhage 8–12 hours
Major surgery Von Willebrand factor 40–70% of the original Occasionally
Major trauma Factor-XIII 20–30% of the original Occasionally
Severe abdominal pain 20 20–25 IU every 12 hours Fibrinogen 150–250 mg Occasionally
Extraction of permanent 20 20 IU every 12 hours Fibronectin 55 mg Occasionally
teeth Often not necessary
75% units sampled and tested should have the values
Tongue or mouth laceration 20 20 IU every 12 hours indicated here.
176  Section 1  Clinical Hemotherapy
Pooling of cryoprecipitate
Cryo is thawed at 30°–37°C in circulating water bath and all
bags are pooled in one bag by means of bag to bag connector
under laminar flow. Wash each empty bag with 10 mL of
sterile normal saline to dissolve residual cryoprecipitate and
add to the pooled cryoprecipitate. It should be used preferably
immediately after thawing and pooling.
Outdate: 6 hours after thaw and 4 hours after pooling.
Potential Adverse Reactions5
Cryoprecipitate administration may be associated with
nonspecific side effects related to its protein, cytokine, and
isohemagglutinin content. Adverse reactions include fever,
chills, and allergic reactions of variable severity.
References
1. American Association of Blood Banks, America’s Blood Centes,
American Red Cross. Circular of information for the use of
human blood and blood components, 2002. pp. 26-8.
2. Saxena S, Odono V, Francis RB Jr, et al. Can storage of thawed
cryoprecipitate be extended to more than six hours? Am J Clin
Pathol. 1990;94:203-6.
3. Makroo RN. Practice of safe blood transfusion. Compendium
of Transfusion Medicine, Blood component preparation and
therapy, 2nd edn; 2009. pp. 171-6.
4. Blood components preparation and their uses; Transfusion
Medicine Technical Manual. Dr RK Saran (Eds), 2nd edn; 2003.
pp. 221-3.
5. Ed Hilluyer CD, Silberstein LE, Ness PM, Aderson KC, Roback
ID. Cryoprecipitate and Related Products, Leon L Su, Lennart E
Logdberg; Blood Banking and Transfusion Medicine. Churchil
Livingstone Philadelphia; 2007. pp. 270-6.
 Section

2
Cellular Therapies
Stem Cell Transplantation:
Brief Overview

Pankaj Malhotra
50
There have been recent advances in the field of stem cell from unrelated donor who has HLA match or when donor and
transplantation especially in the last two decades. This recipient differ by one HLA antigen. There is also increasing
procedure is the only curative option for some of the use of peripheral blood stem cell or progenitor cell transplant
benign as well as malignant hematological disorders. The (PBPC) for allografting. Even in autologous bone marrow
procedure should preferably be called hematopoietic stem transplant (BMT), there is an increasing use of peripheral
cell transplantation (HSCT) because the source of stem blood stem cell (progenitor cell) transplant. The use of PBSC
cells could be from bone marrow, peripheral blood or results in more rapid engraftment, shorter hospital stay and
umbilical cord. HSCT is generally performed for treatment thus decreased medical costs.
of patients with genetic disorders like thalassemia major; Before BMT/PBPC transplantation, patient’s bone
malignant disorders like acute leukemia and in conditions marrow is conditioned with high dose chemotherapy with or
of hypofunctioning of marrow like aplastic anemia. The without total body irradiation. The purpose of conditioning
procedure is called Allogeneic if donor and recipient are is to destroy the recipient marrow including the tumor
of different genetic origin and autologous if patient’s own cells if present. By destroying the cells populating the bone
marrow is transfused (transplanted) after chemotherapy and marrow, twin objectives of creating space for engraftment
or radiotherapy (Table 1). and destroying host immune effector cells to reduce rejection
For Allogeneic BMT, selection of donor is determined by of the allografted stem cells are achieved.
human leukocyte antigen (HLA) matching. HLA is composed The donor stem cells are collected by stem cell apheresis
of closely linked genes on chromosome 6. There is one in technique from peripheral blood or by multiple marrow
four chance of HLA matching between two siblings. The most aspiration from iliac crests. Mononuclear cell count or
widely used transplants are those between HLA-identical preferably CD 34 counts is calculated on these products to
siblings. However, there is now increasing use of marrow know the adequacy of collection. It is then given to recipient
by intravenous infusion (there is no surgery involved in stem
Table 1  Types of hematopoietic stem cell transplantation cell/bone marrow transplantation). The donor’s marrow cells
Source Type Donor almost go exclusively to recipient marrow and start producing
different series of normal cell lines usually within 2–4 weeks.
Bone marrow Allogeneic Sibling
Use of colony stimulating factors (G-CSF, GM-CSF) has
Voluntary donor shortened the period further down to between 1–2 weeks.
Syngeneic Twin sibling However, during this period these patients need utmost
Autologous Self care to prevent complications due to thrombocytopenia
and granulocytopenia. Life-threatening bleeding due to
PBSC* Allogeneic Sibling
thrombocytopenia is prevented by platelet transfusions.
Voluntary donor Granulocytopenia is a major risk factor for bacterial and
Syngeneic Twin sibling fungal infections in immediate post transplant period and
Autologous Self is prevented by appropriate antibiotics and antifungals. All
blood products are given to these patients after irradiation to
Umbilical cord blood Allogeneic Sibling
prevent graft versus host disease.
Unrelated Successful engraftment is signaled by rise in granulocyte
* Peripheral blood stem cell and platelets and appearance of reticulocytes of the donor’s
180  Section 2  Cellular Therapies
marrow origin in the peripheral blood film. Proof of
engraftment depends on use of cytogenetic and other genetic
markers to distinguish donor from host cells.
The complications that follow successful transplant are
graft versus host disease (GvHD) which can be acute or
chronic infections, veno-occlusive liver disease, recurrence
of leukemia and miscellaneous (hemmorrhagic cystitis,
sterility, leukoencephalopathy, cataract, etc.). Immunological
reactions due to immunocompetent cells from donor are not
always harmful. In fact to a large extent, graft versus tumor
effect is thought to be responsible for cures among patients
with malignant disorders.
The success of transplant depends upon the age of the
patient and the disease for which the transplant is performed.
It is more successful in younger age group (less than 40 years)
although allogeneic BMT and PBPC are performed even
after this age. The overall results of BMT and PBPC are still
poor in elderly age group because of decreased tolerability to
conditioning regimen and increased risk of infections.
With improvement in supportive techniques and use of
effective treatment for graft versus host disease; there has
been an improvement in the outcome. The list of indications
for hematopoietic stem cell transplantation has been
expanding. However, it still continues to be a procedure
with fairly high morbidity and mortality. It is hoped that
the results shall improve following better understanding of
transplantation biology as well as better methods for control
and management of various complications.
Suggested Reading
1. Baron F, Storb R. Allogeneic hematopoietic cell transplantation
as treatment for hematological malignancies: a review.
Springer Semin Immunopathol. 2004;26:71-94.
2. Giralt S, Bishop MR. Principles and overview of allogeneic
hematopoietic stem cell transplantation. Cancer Treat Res.
2009;144:1-21.
3. Gyurkocza B, Rezvani A, Storb RF. Allogeneic hematopoietic
cell transplantation: the state of the art. Expert Rev Hematol:
2010;3:285-99.
4. Körbling M, Freireich EJ. Twenty-five years of peripheral blood
stem cell transplantation. Blood. 2011;117:6411-6.
5. Vaughan W, Seshadri T, Bridges M, Keating A. The principles
and overview of autologous hematopoietic stem cell trans­
plantation. Cancer Treat Res. 2009;144:23-45.
6. Wingard JR, Hsu J, Hiemenz JW. Hematopoietic stem cell trans­
plantation: an overview of infection risks and epidemiology.
Infect Dis Clin North Am. 2010;24:257-72.
Stem Cell in Regenerative
Medicine: Applications and
Cautions
RR Sharma
Stem cells are considered the master cells, capable of
both self-renewal and multilineage differentiation. Recent
investigations have identified them as a potentially novel
cell therapy for regenerative medicine largely because of
their ability to differentiate into many functional cell types.
It is anticipated that stem cell therapy could solve many
medical challenges facing humanity, increase our knowledge
of pathogenesis, allow us to screen for new drugs for safety
and effectiveness, and treat a variety of diseases. It is highly
expected that detailed investigations of stem cell biology
and clinical applicability will result in revolutions in medical
technologies. Most early work on stem cells was carried out
with pluripotent embryonic stem (ES) cells derived from inner
cell mass of blastocyst embryo, which, however, introduced
a series of ethical problems in clinical applications. To
avoid such ethical issues and create histocompatibility, new
technologies have enabled tissue cells to become induced
pluripotent stem (iPS) cells.1 One characteristic of ES cells
and iPS cells is their ability to form teratomas, which, in turn,
is a major concern for future clinical application.2,3 Moreover,
it is likely that iPS cells are more tumorigenic than ES cells.4
The teratoma-forming property of stem cells is considered
a major obstacle for biomedicine by the US Food and Drug
Administration (FDA).5
Translation of stem cell science into
regenerative medicine
Regenerative medicine has been proposed to provide
in the near future cures for debilitating diseases such as
diabetes, cardiovascular disease, and Alzheimer’s disease.6
In particular, the use of stem cells to repair damaged
tissues has led to a number of clinical trials in wide range
of disorders.6 However, despite the enormous potential of
stem cells for regenerative medicine, there are many hurdles
to overcome to develop these therapies. Although, we are
able to isolate, culture, expand and differentiate stem cells
in vitro, the requirements for in vivo survival, proliferation
and differentiation into functional tissue, are essentially
51
unknown. In addition, we must understand the role of
immune cells that may reject donor cells or trigger other
immune reactions leading to failure of the therapy or even
worse, death of the patient. Added to these biological issues
are the regulatory requirements for conducting clinical trials
and the complexities of cellular products. Many aspects of
drug development including toxicology, development of
potency assessment, determination of dosing and safety
assessment in animal models are not readily applicable to
cellular products. For example, testing in animal models is
complicated due to the human origin of the clinical cellular
products and xeno rejection in animals. In many cases the
animal equivalent is biologically different and cannot be
used in place of the human cellular product. Scientists have
developed mouse models of immune compromised mice
that enable engraftment of the human cells. However, these
models obviously do not evaluate the immune aspects of
the cellular therapy. So how can Regenerative Medicine be
developed?
As detailed in the perspective article by Daley,7 there are
many clinical trials in progress around the world evaluating
cellular approaches to disease. Many of these studies
involve testing mesenchymal stem cells (MSCs) in a range
of clinical indications. The early results from these trials,
along with extensive animal data, suggest limited integration
of the infused cells into the target tissue and concluding
that the benefits observed may be due to production of
cytokines or other soluble mediators. Further it appears that
benefits obtained from these treatments may be transient
due to the lack of engraftment of the MSCs. As many of
the treatments are invasive procedures, such as catheter
delivery of cells to the heart,8 it is unlikely that multiple
treatments will be feasible. A major challenge will be to
continue with clinical trials when the initial studies show
limited efficacy. Given the history of cellular therapies in
bone marrow transplantation and immunotherapy9,10 it
is only through carefully designed incremental studies that
these approaches will be optimized. Some cellular products
will be developed through commercial entities and the same
182  Section 2  Cellular Therapies
questions will become critical to the long term survival of the
companies with stock holders and the stock market expecting
breakthrough results in clinical trials. Another consideration
relates to the multicellular composition of tissues and organs.
Ischemic events will damage all the cells of the tissue or organ
and so multicellular products will most likely be needed to
restore full capacity. This may require sequential treatments
with an initial cellular product, such as MSCs, to repair the
tissue microenvironment, followed by a second cellular
product to replace the functional mature cells of the tissue.
In the absence of repair to the microenvironment, infusion of
functional cells may result in poor engraftment of survival.
Scientific advances in stem cell biology are being driven by
the current intellectual ferment and excitement of the field,
but when and how these advances will be translated into
successful treatments remain fertile questions for debate.
Will cell therapies remain a highly patient-focused endeavor
performed solely in academic medical centers, akin to bone
marrow or solid organ transplantation? Or will stem cells ever
become commercial, pharmaceutical grade “off-the-shelf”
products?
One might imagine a future in which medical centers offer
highly customized, patient focused approaches to stem cell
treatments, perhaps utilizing the products of personalized
induced pluripotent stem cells. IPS cells have enormous
theoretical appeal as vehicles for combined gene repair and
cell replacement therapy for genetic disease Newer forms
of stem cell transplant could replicate the current status of
bone marrow transplantation, which has developed into a
remarkably complex infrastructure for capturing cellular and
molecular information in international registries for literally
millions of potential donors, and entails lengthy, costly, and
risky interventions in intensive clinical care settings. Given the
imperative of treating patients in need, stem cell transplants
for genetic and acquired diseases will emerge from academic
centers because clinician investigators will develop them
and patients will demand them. Like gene repair (“gene
therapy”), cell replacement therapies will likely serve rare
conditions first, and pertain to small numbers of patients
receiving highly individualized treatments, perhaps coupling
gene repair with autologous cell replacement approaches, for
example for blood diseases. Such small-scale applications
will dominate until and unless generic interventions and off-
the-shelf approaches prove feasible.
The prospects for more widespread stem cell-based
treatments depends on either solving the immune rejection
barrier, through advances in promoting immune tolerance
to allogeneic tissues, or accepting the use of immune
suppression—even lifelong—to facilitate allogeneic cell
therapies. Immune suppression is already standard for organ
transplantation, so we know that its use to facilitate life-
sustaining cell therapy is feasible. Because cell manufacture
is likely to be the most costly and time-consuming aspect
limiting cell therapies, the prospects for realizing economies
of scale would seem to call for the establishment of master
cell banks that could be the source of cells “off-the-shelf”. The
polymorphism of histocompatibility genes and the resulting
variety of tissue types is far too great in human populations
to expect banks to be able to supply perfect tissue matches
for all potential patients. Instead, one might envision banks
of cells derived from donors with highly common genotypes
of the histocompatibility genes. This type of approach would
be greatly facilitated by cell strains with homozygosity of
histocompatibility loci. Past approximations of the number of
cell lines that would be needed in such a repository or master
cell bank, based on modeling data from pools of kidney
transplant patients and recipients in the United Kingdom and
Japan, have suggested that a bank comprised on the order of
10–50 cell lines might effectively provide a single HLA antigen
match (deemed a minimal requirement for acceptable solid
organ transplantation) for approximately 80% of the local
population (Gourraud et al.,11 2012; Nakajima et al.,12 2007;
Taylor et al.,13 2011). While encouraging, these numbers
suggest that some kind of dual system might well be needed
in which the vast majority of individuals can benefit from
off the- shelf therapies, but personalized autologous cells
derived via reprogramming would be needed for those with
difficult-to-match tissue types.
Threats to clinical translation and
to the integrity of regenerative
medicine
Translating the basic discoveries of stem cell biology into
robust, effective, and safe new modalities of care will
mean solving new challenges; before success, regenerative
medicine will suffer many setbacks. While translating too
timidly might deprive needy patients of precious time
and life quality, testing cells in patients before a deeper
understanding of how stem cells work is risky too. We need
to be confident that we understand the full spectrum of safety
concerns and can therefore avoid placing patients at undue
risk. We also need to design rigorous controlled trials where
evidence for clinical efficacy can be defined precisely, rather
than depend upon anecdote and clinical observation alone.
Given that patients and practitioners may carry unrealistic
expectations of clinical efficacy, there is a high likelihood
for a robust placebo effect as well as interpretive bias in
reporting of clinical results. We also need to be conscious of
not exhausting resources that would be better spent on more
practical health care needs. Premature application runs the
risk of high-profile failure that would sully the credibility of
this still-developing field.
With the goal of advancing clinical investigation while
preserving rigor, promoting medical innovation while protecting
patients, and ensuring integrity in regenerative medicine while
respecting autonomy of individual practitioners and patients,
the International Society for Stem Cell Research (ISSCR)
assembled an international group of scientists, surgeons, gene
therapists, bioethicists, patient advocates, and attorneys and
 Chapter 51  Stem Cell in Regenerative Medicine: Applications and Cautions  183
composed “The ISSCR Guidelines for the Clinical Translation of
Stem Cells” (Hyun et al., 2008). These “Guidelines” articulated
principles and standards as a roadmap for practitioners and
regulatory bodies when considering if, when, and how to allow
tests of experimental stem cell therapies in actual patients.
The guidelines call for independent and rigorous analysis of
the decision to test novel treatments in patients, by reviewers
with relevant area-specific expertise, who are free of conflicts
of interest that might lead to positive or negative bias. Expert
judgment about the reliability and rigor of the pre-clinical
evidence for efficacy and safety of cellular products is essential
for weighing the potential risks against the potential benefits
before launching a clinical trial. Because no pre-clinical animal
or cellular model is entirely predictive of outcomes in patients,
a credible and rigorous process of informed consent is essential
to protecting the autonomy of patients and their thoughtful
engagement in the research process, where they consent to
participate without heightened expectations or therapeutic
misconception; such wishful thinking renders patients
vulnerable to exploitation and contaminates interpretations
of therapeutic efficacy. Given the enormous attention given to
stem cells in the media over the last decade, stem cell-based
interventions are certain to elicit a strong placebo effect, which
makes it that much more critical to supplant anecdotal evidence
for clinical efficacy with rigorous, blinded, and randomized
data on patient outcomes.
In summary, although cellular therapy approaches to
regenerative medicine offer great potential for resolving
debilitating diseases, we need to increase our basic
understanding of the biology of tissue structure and the
cellular make up of tissues and organs. Carefully designed
clinical trials will be needed to move forward in what will
most likely be a step wise learning curve. These clinical trials
will need to be performed in a safe manner and adhere to the
regulatory framework of the FDA or regulatory agencies in
different parts of the world.
References
1. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent
stem cell lines derived from human somatic cells. Science.
2007;318: 1917-20.
2. Wong DJ, Liu H, Ridky TW, et al. Module map of stem cell genes
guides creation of epithelial cancer stem cells. Cell Stem Cell.
2008;2:333-44.
3. Miura K, Okada Y, Aoi T, et al. Variation in the safety of induced
pluripotent stem cell lines. Nat Biotechnol. 2009;27:743-5.
4. Gutierrez-Aranda I, Ramos-Mejia V, Bueno C, et al. Human
induced pluripotent stem cells develop teratoma more effi-
ciently and faster than human embryonic stem cells regardless
the site of injection. Stem Cells. 2010;28:1568-70.
5. Fink DW Jr. FDA regulation of stem cellbased products. Science.
2009;324:1662-3.
6. A New Vision – A Future of Regenerative Medicine. US.
Department of Health and Human Services Washington, DC.
7. Daley GQ. The promise and perils of stem cell therapeutics.
Cell Stem Cell. 2012;10:740-9.
8. Williams AR, Trachtenberg B, Velazquez DL, McNiece I,
Altman P, et al. Intramyocardial stem cell injection in patients
with ischemic cardiomyopathy: Functional recovery and
reverse remodeling. Circ Res. 2011:108:792-6.
9. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J
Med. 2006;354:1813-26.
10. Larenas Linnemann DE . One hundred years of immunotherapy:
review of the first landmark studies. Allergy Asthma Proc. 2012;
33:122-8.
11. Gourraud PA, Gilson L, Girard M, Peschanski M. The role of
human leukocyte antigen matching in the development of
multiethnic “haplobank” of induced pluripotent stem cell
lines. Stem Cells. 2012; 30:180-6.
12. Nakajima F, Tokunaga K, Nakatsuji N. Human leukocyte
antigen matching estimations in a hypothetical bank of human
embryonic stem cell lines in the Japanese population for use in
cell transplantation therapy. Stem Cells. 2007;25:983-5.
13. Taylor CJ, Bolton EM, Bradley JA. Immunological considerations
for embryonic and induced pluripotent stem cell banking.
Philos Trans R Soc Lond B Biol Sci. 2011;366:2312-22.
Stem Cell Therapy in
Cardiovascular Diseases
Hamed Bashir, Ajay Bahl, KK Talwar
Introduction
Stem cells are a class of undifferentiated cells that are able
to differentiate into specialized cell types. Commonly, stem
cells come from two main sources—embryonic stem cells
and adult tissue (adult stem cells).
Embryonic Stem Cell
Embryonic stem (ES) cells are derived from the inner cell
mass of the blastocyst-stage embryo, late in the first week
after fertilization. They are considered to be pluripotent, able
to give rise to many different cell lineages.
Adult stem cells
Adult or somatic stem cells exist throughout the body after
embryonic development and are found inside of different
types of tissue. These stem cells have been found in tissues
such as the brain, bone marrow, blood, blood vessels, skeletal
muscles, skin, and the liver. They remain in a quiescent
or non-dividing state for years until activated by disease
or tissue injury. Adult stem cells can divide or self-renew
indefinitely, enabling them to generate a range of cell types
from the originating organ or even regenerate the entire
original organ. It is generally thought that adult stem cells are
limited in their ability to differentiate based on their tissue
of origin, but there is some evidence to suggest that they can
differentiate to become other cell types.
Mechanism of Action of Stem Cells
Several theories have been proposed regarding the
mechanism of action of stem cells. It is believed that delivery
of the appropriate stem cells would repair a damaged heart
via active myocardial regeneration resulting from trans-
differentiation of the administered stem cells.1-4 Exogenous
stem cells may also stimulate proliferation of endogenous
cardiac precursors or stem cells through neovascularization5or
52
paracrine signalling actions.6 Observations in preclinical
and clinical scenarios that all of these events occur allow us
to generate a new concept that cellular therapy contributes
to the restoration of stem cell niches, facilitating the ability
of the heart to heal itself.7 Other proposed mechanisms
include cardiac repair via fusion of donor cells with host
cardiomyocytes.8 Some investigators suggest that the effects
of stem cells are mediated by altering mechanical properties
to strengthen the MI scar, thereby preventing deterioration in
cardiac function.9
Types of Cells Contemplated for
Cellular Regenerative Therapy
Embryonic Stem Cell
Embryonic stem (ES) cells are derived from the inner cell
mass of the blastocyst-stage embryo, late in the first week
after fertilization. They are considered to be pluripotent, able
to give rise to many different cell lineages. There is a growing
body of knowledge from animal models regarding the steps
of isolation, differentiation, and clinical application. ES cells
have not been used clinically for cardiac regenerative therapy
mainly because of ethical issues and the risk of teratoma
formation.
Resident Cardiac Stem Cells
In recent years, compelling evidence has accumulated
suggesting that the heart has endogenous regenerative
potential. Recent studies have isolated undifferentiated
cells that are clonogenic, express stem and endothelial
progenitor cell (EPC) antigens/markers, and appear to have
the properties of adult cardiac stem cells.10,11 These cells most
likely mediate endogenous mechanisms for minor repair and
for replacement of ongoing cell turnover within the adult
heart. More importantly, they may represent a therapeutic
target that, if enhanced, could induce cardiac self-repair.
 Chapter 52  Stem Cell Therapy in Cardiovascular Diseases  185
Skeletal Myoblasts
Autologous skeletal myoblasts are another potential source
for cardiac repair because of their biological properties
and lack of ethical and immunological problems. Skeletal
myoblasts or satellite cells are the reservoir of regenerative
cells for skeletal muscle tissue; they have the ability for self
renewal and differentiation if case of muscle injury.12 They
have many desirable features as donor cells, including the
ability to be amplified in an undifferentiated state in vitro
and high resistance to tissue ischemia. They also have been
shown to continue proliferation in vivo to a certain extent,
which gives rise to a larger graft size. However, they do not
form gap junctions. Therefore, they lack electromechanical
coupling and do not synchronously contract with the host
myocardium. This can cause risk of arrhythmias.
Human Adult Bone Marrow
Derived Stem Cells
The observation that bone marrow elements contribute to
cardiac repair in the infarcted heart served as the rationale for
adult bone marrow cell therapy after myocardial infarction.
Jackson and co-workers transplanted wild-type mice with
green fluorescentprotein (GFP)–positive bone marrow
and then induced MI through coronary artery ligation
and reperfusion.13 They demonstrated that bone marrow
elements contributed to cardiomyocyte and endothelial
cell formation after myocardial infarction by finding GFP-
positive cardiomyocytes and endothelial cells. Bone marrow
mononuclear cells (BMMNCs) have been most frequently
used in clinical trials.
Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are found in bone marrow,
muscle, skin, and adipose tissue and are characterized by
the potential to differentiate into any tissue of mesenchymal
origin, including muscle, fibroblasts, bone, tendon, ligament,
and adipose tissue.14 MSCs from adult bone marrow can be
separated by density gradient centrifugation and adhering-
cell culture in defined serum-containing medium.
Endothelial Progenitor Cells
Cells with phenotypic and functional characteristics similar
to the fetal angioblast also are present in adult human
bone marrow. These cells, known as EPCs, express some,
but not all, cell surface markers characteristic of mature
endothelium, certain surface markers of hematopoietic cells,
and transcription factors that identify them as precursor cells.
Methods of Stem Cell Delivery
A major goal of cardiac stem cell therapy is to transplant
enough cells into the myocardium at the site of injury or
infarction to maximize restoration of function. Several
different approaches currently are being used to deliver stem
cells.
Transvascular Route
A transvascular approach is particularly well suited to treat
patients with acutely infarcted and reperfused myocardium.
Stem cells can be infused directly into the coronary arteries
and have a greater likelihood of remaining in the injured
myocardium as a result of the activation of adhesion molecules
and chemokines. The advantage of an intracoronary infusion
is that the cells are directed to a particular territory. An
alternative approach is to inject stem cells intravenously. In
the setting of myocardial infarction, circulating stem cells
have been shown to home to sites of injury, but the number
of cells that home to the heart in this way is significantly less
than by local injection.
Direct Injection into the Ventricular Wall
Direct injection of stem cells is used in patients presenting
with established cardiac dysfunction in whom a transvascular
approach may not be possible because of total occlusion or
poor flow within the vessel of the affected territory. There
are three different approaches to direct injection. A trans-
endocardial approach can be used in which a needle catheter
is advanced across the aortic valve and positioned against the
endocardial surface. Cells can then be injected directly into
the left ventricle. Electrophysiological mapping can be used to
differentiate sites of viable, ischemic, or scarred myocardium.
In a trans-epicardial approach, cells are injected during
open heart surgery. The advantage of this approach is that
it allows direct visualization of the myocardium and easier
identification of regions of scar and border zones of infarcted
tissues. A third approach involves the delivery of cells through
one of the cardiac veins directly into the myocardium. The
limitation of this approach is that positioning the catheter
within a particular coronary vein may be considerably more
time consuming and technically challenging.
Stem Cell Therapy for Cardiac Repair
Coronary heart disease and heart failure continue to be
significant burdens to healthcare systems. Despite recent
advances in medical and device therapy for heart failure,
the incidence, hospitalization, and mortality rates continue
to rise. Left ventricular systolic dysfunction, a major
determinant of prognosis, is associated with significant
loss of cardiomyocytes which was previously thought to be
irreversible as the heart was considered a post-mitotic organ.
The heart has been considered a terminally differentiated
organ lacking self-renewal capabilities but this dogma has
been challenged recently, particularly with the demonstration
of continued cell division within the adult heart following
injuries such as myocardial infarction. Several independent
186  Section 2  Cellular Therapies
investigators have now isolated and identified resident cardiac
stem cells which are capable of differentiating into multiple
cardiac cell lineages, such as cardiomyocytes and vascular
smooth muscle cells.15,16 These cells have been studied in
animal models of myocardial infarction with beneficial
outcomes in terms of reducing infarct size and improving
LV function.17Resident cardiac stem cells, however, have
a low mitotic rate. To increase these cells clinically useful,
harvesting technique need to be perfected.
The possibility of using stem cell-based therapies for people
suffering an acute MI or living with CHF has captured the
imagination of both the medical and popular communities.
The publication of Menasche et al.18 describing the first
patients to receive skeletal myoblasts spawned a profusion of
small clinical studies investigating cellular therapy for cardiac
repair.
Results of Stem cell transplant
in clinical trials
Clinical trials have so far focused on three main situations:
acute myocardial infarction (with the hope of preventing
left ventricular systolic dysfunction), chronic heart failure
secondary to previous myocardial infarction and DCM.
Stem Cell Therapy in Acute
Myocardial Infarction
Four main randomized controlled trials (RCTs) have been
published with positive findings so far. The TOPCARE-
AMI trial was the first published RCT to demonstrate the
potential beneficial effect of BSMC therapy following AMI
with improvement in the LVEF from 51.6+9.6 to 60.1+8.6%
at 4 months.19 In the BOOST trial global LVEF improved by
6% at 6 months. This improvement in LVEF was transient
and was not sustained at 18 months and 5 years follow-up.
In this trial, improvement was maintained only in patients
with larger transmural infarcts at long-term follow-up.20,21
Patients in the BOOST trial had relatively preserved left
ventricular functions with baseline mean LVEF being over
50%. Improvement in LVEF seen in the sub-group of patients
with larger infarcts needs confirmation in a larger study. In the
REPAIR-AMI trial, 22 the largest trial to date, BMSC therapy
was associated with an increase in the LVEF of 2.8% at 12
months and the FINCELL trial23 reported an improvement of
5%. In contrast, three RCTs have produced neutral findings
Janssens et al.24 from the LEUVEN-AMI study, reported no
changes in global LVEF after BMSC infusion, although sub-
set analysis showed possible benefits in reducing infarct size
in patients who suffered the largest infarcts. In the ASTAMI
trial, BMSC administration had no significant effect on the
LVEF, LV volumes, or infarct size.25 Finally, in the recently
published HEBE trial, no changes in global or regional LV
systolic function were reported after BMSC therapy.26 A
large randomized multi-center trial enrolling 250 Indian
patients using bone marrow stem cells in large anterior wall
myocardial infarction has been completed, however, the
results are not yet published.
Stem Cell Therapy in Chronic
Ischemic Heart Failure
The MAGIC trial was the first randomized placebo-controlled
study of myoblast transplantation in patients with LVSD
secondary to previous myocardial infarction and who
required coronary surgery.27 Cells were injected into the
epicardium within scarred areas during open heart surgery.
This study was prematurely stopped as cell injection did not
improve regional or global left ventricular function but led
to a higher number of arrhythmic events. More recently,
percutaneous transcathetra intra myocardial injection of
skeletal myoblasts (CAUSMIC study) into areas of viable
myocardium in patients with severe ischemic heart failure
has shown promise with improvement in NYHA functional
class, quality-of-life and evidence of reverse ventricular
remodelling when compared with controls after 1-year
follow-up.28 In a similar study design the SEISMIC trial
confirmed the safety and feasibility of catheter-based intra
myocardial injection of skeletal myoblasts. There appeared
to be some improvement in patient symptoms but the study
failed to show any significant improvement in the LVEF.
BMSC transplantation has also been assessed in patients
with chronic ischemic heart failure and has been delivered
either directly (transepicardial injection) during CABG
surgery or by percutaneous intracoronary injection. One
of the initial surgical studies injected unmanipulated BM,
obtained from a sternal aspirate, directly into areas of scarred
myocardium in 14 patients undergoing CABG. There appeared
to be an improvement in regional contractility in those
segments treated with revascularization and cell injection.
However, a follow-up randomized controlled trial, with 63
patients undergoing elective CABG, by the same group failed
to show any benefit of BMSC injection. In contrast to this,
another randomized study with a similar study design (40
patients undergoing CABG) showed additional cell injection
was associated with better global left ventricular function
than CABG alone at 6 months. In the TOPCARE-CHD trial,
intracoronary delivery of BMSCs showed an improvement in
the LVEF of 2.9% with no major adverse cardiac events.29
Stem Cell Therapy in Dilated Cardiomyopathy
A few small studies have been carried out using BMSCs
and autologous CD34 positive stem cells injected by the
intracoronary route in DCM patients. These studies have
consistently shown a modest improvement in LVEF of 3–8%
which is sustained up to 5 years.30-33An Indian study showed
an improvement of LVEF by 5.9% in the stem cell group
at 5 years follow-up. In addition improvement in exercise
capacity and fall in brain natriuretic peptide levels also
 Chapter 52  Stem Cell Therapy in Cardiovascular Diseases  187
occurs. These pilot studies have prompted the design of large
RCTs to confirm these findings including the Regenerate-
DCM study which recently began recruiting patients. This is
the first randomized, double blind, placebo-controlled trial
worldwide investigating the role of G-CSF and BMSCs to
improve cardiac function in patients with DCM.34
Concerns
So far several clinical studies exploring the safety and
feasibility of stem cell therapy have been conducted.
These studies have used a myriad of different cell types
and preparations, each in a small number of patients with
different disease states. Present clinical evidence suggests
that stem cell therapy might work, though the efficacy is
modest at best. Initial clinical studies have generated a great
deal of hope, we should take into account the lessons learned
from the translation of therapeutic angiogenesis into clinical
studies, where great expectations raised by open studies have
not been confirmed by subsequent randomized trials.
Although studies are underway, fundamental questions
need to be addressed experimentally. What is the fate of the
injected cells after transplantation? How long do they survive?
Do the cells incorporate, or is transient retention sufficient to
promote functional effects? Genetic and transgenic markers
should be used to determine the lineage commitment of
engrafted cells.
Conclusion
• Stem cell therapy is an exciting novel treatment strategy for
the management of patients with left ventricular systolic
dysfunction.
• The efficacy of cell therapy in improving cardiac
function has been proved in animal models although the
mechanisms of action need to be clearly defined.
• Clinical trials of cell therapy have been performed in the
settings of acute myocardial infarction, chronic ischemic
heart failure and dilated cardiomyopathy. These trials have
uniformly shown the safety of this treatment regardless of
study design. Unfortunately clinical trials have shown at
best only modest improvement in left ventricular function.
• The clinical efficacy of cell therapy remains to be fully
translated and needs far more basic research and closer
collaboration between scientists and clinicians.
References
1. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells
regenerate infarcted myocardium. Nature. 2001;410:701-5.
2. Condorelli G, Borello U, De Angelis L, et al. Cardiomyocytes
induce endothelial cells to transdifferentiate into cardiac
muscle: implications for myocardium regeneration. Proc Natl
Acad Sci USA. 2001;98:10733-8.
3. Kudo M, Wang Y, Wani MA, et al. Implantation of bone marrow
stem cells reduces the infarction and fibrosis in ischemic
mouse heart. J Mol Cell Cardiol. 2003;35:1113-9.
4. Olivares EL, Ribeiro VP, Werneck de Castro JPS et al. Bone
marrow stromal cells improve cardiac performance in healed
infarcted rat hearts. Am J Physiol Heart Circ Physiol. 2004;287:
H464-H70.
5. Schuster MD, Kocher AA, Seki T, et al. Myocardial neovascular-
ization by bone marrow angioblasts results in cardiomyocyte
regeneration. Am J Physiol Heart Circ Physiol. 2004;287:H525-
H32.
6. Gnecchi M, He H, Liang OD, et al. Paracrine action accounts
for marked protection of ischemic heart by Akt-modified
mesenchymal stem cells. Nat Med. 2005;11:367-8.
7. Moore KA, Lemischka IR. Stem cells and their niches. Science.
2006; 311:1880-85.
8. Nygren JM, Jovinge S, Breitbach M, et al. Bone marrow-
derived hematopoietic cells generate cardiomyocytes at a low
frequency through cell fusion, but not transdifferentiation. Nat
Med. 2004;10:494-501.
9. Fujii T, Yau TM, Weisel RD, Ohno N, Mickle DAG, Shiono N,
Ozawa T, et al. Cell transplantation to prevent heart failure: a
comparison of cell types. Ann Thorac Surg. 2003;76:2062-70.
10. Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem
cells are multipotent and support myocardial regeneration.
Cell. 2003;114:763-6.
11. Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells
from adult myocardium: homing, differentiation, and fusion
after infarction. Proc Natl Acad Sci USA. 2003;100:12313-8.
12. Kuethe F, Figulla HR, Herzau M, et al. Treatment with
granulocyte colony-stimulating factor for mobilization of bone
marrow cells in patients with acute myocardial infarction. Am
Heart J. 2005;150:115.
13. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic
cardiac muscle and vascular endothelium by adult stem cells.
J Clin Invest. 2001;107:1395-1402.
14. Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9:641-
50.
15. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human
cardiac myocytes divide after myocardial infarction. N Eng J
Med. 2001;344:1750-7.
16. Messina E, De AL, Frati G, et al. Isolation and expansion of
adult cardiac stem cells from human and murine heart. Circ
Res. 2004;95:911-21.
17. Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells
from adult myocardium: homing, differentiation, and fusion
after infarction. Proc Natl Acad Sci USA. 2003;100:12313-8.
18. Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M,
Duboc D, Schwartz K, et al. Myoblast transplantation for heart
failure. Lancet. 2001;357:279-80.
19. Assmus B, Schächinger V, Teupe C, et al. Transplantation
of Progenitor Cells and Regeneration Enhancement in
Acute Myocardial Infarction (TOPCARE-AMI). Circulation.
2002;106:3009-17.
20. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous
bone-marrow cell transfer after myocardial infarction: the
BOOST randomised controlled clinical trial. Lancet. 2004;
364:141-8.
21. Meyer GP, Wollert KC, Lotz J, et al. Intracoronary bone marrow
cell transfer after myocardial infarction: 5-year follow-up from
the randomized-controlled BOOST trial. Eur Heart J. 2009;
30:2978-84.
22. Schächinger V, Erbs S, Elsässer A, et al. REPAIR-AMI Investi-
gators. Intracoronary bone marrow-derived progenitor cells in
acute myocardial infarction. N Eng J Med. 2006;355:1210-21.
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23. Huikuri HV, Kervinen K, Niemela M, et al. Effects of
intracoronary injection of mononuclear bone marrow cells on
left ventricular function, arrhythmia risk profile, and restenosis
after thrombolytic therapy of acute myocardial infarction. Eur
Heart J. 2008;29:2723-32.
24. Janssens S, Dubois C, Bogaert J, et al. Autologous bone
marrow derived stem-cell transfer in patients with ST-segment
elevation myocardial infarction: double-blind, randomised
controlled trial. Lancet. 2006;367:113-21.
25. Lunde K, Solheim S, Aakhus S, et al. Intracoronary injection
of mononuclear bone marrow cells in acute myocardial
infarction. N Eng J Med. 2006;355:1199-209.
26. Hirsch A, Nijveldt R, van der Vleuten PA, et al. Intracoronary
infusion of mononuclear cells from bone marrow or peripheral
blood compared with standard therapy in patients afteracute
myocardial infarction treated by primary percutaneous
coronary intervention: results of the randomized controlled
HEBE trial. Eur Heart J. 2011;32:1736-47.
27. Menasche P, Alfieri O, Janssens S, et al. The Myoblast
Autologous Grafting in Ischemic Cardiomyopathy (MAGIC)
trial: first randomized placebo-controlled study of myoblast
transplantation. Circulation. 2008;117:1189-200.
28. Dib N, Dinsmore J, Lababidi Z, et al. One-year follow-up of
feasibility and safety of the first U.S., randomized, controlled
study using 3-dimensional guided catheter-based delivery of
autologous skeletal myoblasts for ischemic cardiomyopathy
(CAuSMIC study). JACC Cardiovasc Interv. 2009;2:9-16.
29. Assmus B, Honold J, Schachinger V, et al. Transcoronary
transplantation of progenitor cells after myocardial infarction.
N Eng J Med. 2006;355:1222-32.
30. Fischer-Rasokat U, Assmus B, Seeger FH, et al. A pilot trial
to assess potential effects of selective intracoronary bone
marrow-derived progenitor cell infusion in patients with
nonischemic dilated cardiomyopathy: final 1-year results of the
transplantation of progenitor cells and functional regeneration
enhancement pilot trial in patients with nonischemic dilated
cardiomyopathy. Circ Heart Fail. 2009;2:417-23.
31. Seth S, Bhargava B, Narang R, et al. The ABCD (Autologous
Bone Marrow Cells in Dilated Cardiomyopathy) trial a long-
term follow-up study. J Am Coll Cardiol. 2010;55:1643-4.
32. Bocchi EA, Bacal F, Guimarães G, et al. Granulocyte-colony
stimulating factor or granulocyte-colony stimulating factor
associated to stem cell intracoronary infusion effects in non
ischemic refractory heart failure. Int J Cardiol. 2010;138:94-7.
33. Vrtovec B, Poglajen G, Lezaic L, et al. Effects of intracoronary
CD34+ stem cell transplantation in nonischemic dilated
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165-73.
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Stem Cell Therapy for
Diabetes Mellitus
Rajesh Rajput
Introduction
Diabetes is a group of diseases characterized by abnormally
high levels of the glucose in the bloodstream. This uncontrolled
hyperglycemia is responsible for most of the microvascular
and macrovascular complications seen in diabetes. Broadly
diabetes is classified into Type 1 diabetes (T1DM) and Type
2 diabetes (T2DM). T1DM typically affects children and
young adults and is characterized by immune mediated
destruction of pancreatic beta cell, as a result all patients with
T1DM needs insulin therapy for proper management of their
disease. T2DM the more common type of disease is seen in
around 90% of adults and tends to affect older, sedentary,
and overweight individuals with a family history of diabetes.
T2DM results from combination of insulin resistance and
relative insulin deficiency and needs combination of diet,
exercise, and oral medication. But a good number of patients
with increasing duration of T2DM ultimately need insulin
therapy for control of hyperglycemia. Despite the fact that
large number of drugs available for management of diabetes,
there is currently no permanent cure for diabetes and large
number of patients need different type of complex insulin
regimen which puts them at risk of hypoglycemia and weight
gain.
It is estimated that by year 2013, approximately 382
million people worldwide are suffering from diabetes out
of which 90% suffers from type 2 diabetes. This number is
expected to rise to 592 million by 2035. Statistics shows that
in year 2012 and 2013, diabetes resulted in 1.5 to 5.1 million
deaths per year making it as 8th leading cause of death and
each year, diabetes affects more people and causes more
deaths than breast cancer and AIDS combined. For decades,
diabetes researchers have been searching for ways to
replace the insulin-producing cells of the pancreas that are
destroyed by a patient's own immune system. To overcome
barriers associated with use of insulin therapy and provide
patients chance of permanent cure of diabetes, every year,
approximately 1,300 people with type 1 diabetes receive
53
whole-organ pancreas transplants. A pancreas transplant can
be carried out in three ways:
1. Simultaneous kidney pancreas transplant (SPK):  Both
the pancreas and kidneys are transplanted together, from
the same donor. This is the most common type, accounting
for nine out of 10 transplants. It is used in people who have
kidney disease as a result of type 1 diabetes.
2. Pancreas after kidney transplant (PAK):  A person first
receives a kidney transplant from a living or deceased
donor. This is then followed by a pancreas transplant from
a deceased donor.
3. Pancreas alone transplant (PTA): Only the pancreas
is transplanted. This is a treatment for patients with very
poorly controlled type 1 diabetes who have hypoglycemic
attacks without warning, and which may threaten their
life.
To prevent the body from rejecting the transplanted
pancreas, patients must take powerful immunosuppressive
drugs for their entire lives, a regimen that makes them
susceptible to a host of other diseases. However, the demand
for transplantable pancreas outweighs their availability and
because of this attempts have been made to cure diabetes by
injecting patients with pancreatic islet cells—the cells of the
pancreas that secrete insulin and other hormones. However,
the requirement for steroid immunosuppressant therapy
to prevent rejection of the cells increases the metabolic
demand on insulin-producing cells and eventually they may
exhaust their capacity to produce insulin. The deleterious
effect of steroids is greater for islet cell transplants than for
whole-organ transplants. As a result, less than 8% of islet cell
transplants performed before last year had been successful.
More recently, James Shapiro and his colleagues in
Edmonton, Alberta, Canada, have developed an experimental
protocol for transplanting islet cells that involves using a
much larger amount of islet cells and a different type of
immunosuppressant therapy that avoids use of steroids.
Despite advances in immunosuppressive regimens, many
hurdles still remain in using this approach on a wide scale to
190  Section 2  Cellular Therapies
treat diabetes. First, donor tissue is not readily available and
islet cells used in transplants are obtained from cadavers, and
the procedure requires at least two cadavers per transplant.
The islet cells must be immunologically compatible, and the
tissue must be freshly obtained—within eight hours of death.
Because of the shortage of organ donors, these requirements
are difficult to meet and the waiting list is expected to far
exceed available tissue, especially if the procedure becomes
widely accepted and available. Further, islet cell transplant
recipients face a lifetime of immunosuppressant therapy,
which makes them susceptible to other serious infections and
diseases. These challenges opened a new era of utilizing the
option of stem cells for treatment of diabetes.
Stem Cell Therapy for Diabetes
Stem cells are self-renewing, unspecialized cells that give
rise to multiple specialized cell types through a process of
differentiation. Theoretically, stem cells could be induced
in the laboratory to become any specialized body cell type,
including pancreatic islet cells, and then transplanted
back into a patient to replace diseased or damaged tissue.
Transplantation of stem cells or stem-cell derived tissue
into human patients has not yet reached the clinical level,
however, and many questions remain regarding the safety
and efficacy of such therapies. Various types of stem cells
with potential to treat diabetes mellitus include:
Source Type of stem cells
Embryonic stem cells Blastocyst-derived embryonic stem
cells
Induced pluripotential stem cells
Umbilical cord stem cells Cord blood hematopoietic stem cells
Cord blood or cord wall mesenchymal
stem cells
Embryonic-like stem cells
Adult stem cells Hematopoietic stem cells
Mesenchymal stem cells
Pancreatic cells Pancreatic stem cells
Reprogrammed adult pancreatic
exocrine cells
In order to better gauge the therapeutic potential of
stem cells, much basic research and animal model testing
is currently being conducted. Scientists have focused on
stem cells from two sources: early-stage embryos and stem
cells from adult tissue. Embryonic and adult stem cells have
different characteristics and are thought to have different
developmental potential. Embryonic stem cells are derived
from the inner cell mass of blastocysts approximately five
days after fertilization in vitro, and appear to have the greatest
therapeutic potential. These cells grow indefinitely in the lab
and are termed pluripotent, as they can develop into any of
the cell types of the body. Scientists have identified specific
growth inhibitors that induce mouse embryonic stem cells
to adopt, in vitro, various characteristics of pancreatic islet
cells, including insulin production, glucose-dependent
insulin release and formation of islet-like aggregates.
Also, when transplanted into diabetic mice, these islet-
like clusters significantly improve diabetic symptoms.
The exact pathways that embryonic stem cells take to
become pancreatic cells are yet to be identified, however,
and embryonic stem cells themselves present a number
of scientific challenges. In general, embryonic stem cells
are hard to maintain in culture in their undifferentiated
state. Because of their uncontrolled growth, embryonic
stem cells may have tumor-forming potential if not fully
differentiated before transplantation into patients. There is
also the risk of immune rejection following transplantation,
so patients receiving the transplants would likely have to
be maintained on immune-suppressant medication.
Adult stem cells, specifically autologous stem cells which
are derived from mature somatic tissues, may avoid the issue
of immune rejection but present distinct challenges of their
own. Adult stem cells do not grow indefinitely in the lab and,
although they can be found in a great number of tissues,
these special cells are generally rare and hard to identify.
In addition, adult stem cells are thought to have a more
limited differentiation profile than embryonic cells, with only
the capacity to differentiate into the specialized cells of the
tissue from which they were derived (e.g. a brain stem cell
could become a neuron but not a blood cell). Preliminary
studies in which adult stem cells of non-pancreatic origin
differentiated to become islet-like cells suggest that
adult stem cells may have greater plasticity, or ability
to develop into a greater number of cell types than previously
believed. In one case, scientists demonstrated that bone
marrow-derived cells transplanted into lethally irradiated
mice are able to differentiate into functional, insulin-
producing islet cells in the pancreases of the recipients.
It is not known, however, whether these differentiated
cells were the progeny of hematopoietic stem cells
(which normally do not give rise to pancreatic cells) or
pancreatic stem cells that happen to reside in the bone
marrow. In another study adult hepatic oval cells, stem
cells of the liver that do not normally give rise to pancreatic
tissue, were shown to differentiate into pancreatic islet-like
clusters in vitro that are able to reverse diabetic symptoms in
mice upon transplantation.
Progressive and inexorable β-cell dysfunction is the
hallmark of type 2 diabetes mellitus (T2DM) and β-cell
regeneration using stem cell therapy may prove to be an
effective modality. Bhansali et al. in their study involved a
total of 10 patients (8 men) with T2DM for >5 years who failed
on triple oral antidiabetic drugs, were on insulin (≥ 0.7 U/
kg/day) at least for 1 year, and glutamic acid decarboxylase
antibody negative. Patients on stable doses of medications
for past 3 months were recruited. Primary end points were
 Chapter 53  Stem Cell Therapy for Diabetes Mellitus  191
reduction in insulin requirement by ≥50% and improvement
in glucagon-stimulated C-peptide levels at the end of 6 months
of autologous hematopoietic stem cell transplantation
(HSCT), while secondary end points were a change in weight
and HbA1c and lipid levels as compared to baseline. Seven
patients were responders and showed a reduction in insulin
requirement by 75% as compared to baseline. Mean duration
to achieve the primary objective was 48 days. Three patients
were able to discontinue insulin completely, although it
was short-lived in one. Mean HbA1c reduction was 1% and
3 of the 7 responders had HbA1c value <7%. A significant
weight loss of 5.5 kg was noted in the responders, whereas,
non responders gained 2.2 kg of weight. However, weight
loss did not correlate with reduction in insulin requirement
(r = 0.68, p = 0.06). There was a significant improvement in
both fasting and glucagon-stimulated C-peptide level in
the group (p = 0.03) and responders (p = 0.03). HOMA-B
increased significantly in the whole group (p = 0.02) and
responders (p = 0.04) whereas, HOMA-IR did not change
significantly (p = 0.74). Reduction in insulin doses correlated
with stimulated C-peptide response at the baseline (r = 0.83,
p = 0.047) and mononuclear cell count of infused stem cells
(r = 0.57, p = 0.04). No serious adverse effects were noted.
Our observations indicate that SCT is a safe and effective
modality of treatment to improve β-cell function in patients
with T2DM. However, further large-scale studies are needed
to substantiate these observations.
Autologous HSCT had been tried across the globe by
various researchers for treatment of T1DM. It has been
observed that high dose immunosuppression followed by
HSCT shows better results among other immunotherapeutic
treatments for the disease as patients with adequate beta
cell reserve achieve insulin independence. However,
this response is not maintained and as seen with T2DM
reoccurrence of the disease is a major challenge with use of
HSCT in future to prevent pr control relapse of T1DM.
Another area of concern while utilizing stem cells for
treatment of diabetes is that it is not clear whether it will
be desirable to produce only beta cells—the islet cells that
manufacture insulin—or whether other types of pancreatic
islet cells are also necessary. Studies by Bernat Soria and
colleagues, for example, indicate that isolated beta cells—
those cultured in the absence of the other types of islet cells—
are less responsive to changes in glucose concentration
than intact islet clusters made up of all islet cell types.
Islet cell clusters typically respond to higher-than-normal
concentrations of glucose by releasing insulin in two phases:
a quick release of high concentrations of insulin and a slower
release of lower concentrations of insulin. In this manner the
beta cells can fine-tune their response to glucose. Extremely
high concentrations of glucose may require that more insulin
be released quickly, while intermediate concentrations of
glucose can be handled by a balance of quickly and slowly
released insulin. Isolated beta cells, as well as islet clusters
with lower-than-normal amounts of non-beta cells, do not
release insulin in this biphasic manner. Instead insulin is
released in an all-or-nothing manner, with no fine-tuning
for intermediate concentrations of glucose in the blood.
Therefore, many researchers believe that it will be preferable
to develop a system in which stem or precursor cell types
can be cultured to produce all the cells of the islet cluster
in order to generate a population of cells that will be able to
coordinate the release of the appropriate amount of insulin to
the physiologically relevant concentrations of glucose in the
blood.
Ethical Issues with use of Stem Cells
The main ethical concern with stem cell research is
balancing the pursuit of a potential therapy for diabetics
with the proper respect for and protection of human life,
an immediate issue when considering the destruction of
living human embryos that is involved in the derivation
of embryonic stem cells. An embryo is a unique human
entity whose biological identity is determined from the
moment of fertilization; this identity is unchanging through
any subsequent differentiation and growth. Natural law,
or those rules for action that all can know through reason
and observation of the world, states that all human persons
possess certain basic rights including the right to life and
physical integrity. Because these rights, or justifiable claims
on others, flow from each human person's intrinsic dignity
as sharers in a common human nature, all living human
beings must be regarded as persons and treated in a morally
equivalent manner. If the human embryo is a person,
then he or she cannot be used as a means toward any end,
however good. No projected therapeutic good that may
come from the use of embryonic stem cells can outweigh
the immediate harm (death) to the human embryo when its
stem cells are extracted. Therefore, research that involves
the derivation of stem cells from living human embryos is
morally unacceptable, even if the projected benefits can be
gotten in no other way. The creation of "surplus" embryos
that one is not planning to implant, a routine part of in vitro
fertilization procedures, must also be rejected.
Other scientifically and ethically viable avenues of stem
cell research do exist that appear to hold great promise for
the eventual treatment of diabetes, including the use of adult
stem cells, and these should be pursued. Although much
basic research remains to be done, adult stem cells appear to
have great therapeutic potential and may even have an edge
over embryonic cells, in that they avoid issues of immune
rejection.
Suggested Reading
1. Anil Bhansali, Vimal Upreti, et al. Stem Cells and Development.
2009;18(10):1407-16.
2. Congregation for the Doctrine of the Faith. Donum Vitae.
Boston: St. Paul Editions, 1987;I:1-5.
192  Section 2  Cellular Therapies
3. Hori Y, et al. Growth inhibitors promote differentiation
of insulin-producing tissue from embryonic stem cells.
Proceedings of the National Academy of Sciences. USA. 2002;
99:16105-10.
4. Ianus A, et al. In vivo derivation of glucose-competent
pancreatic endocrine cells from bone marrow without evidence
of cell fusion. Journal of Clinical Investment. 2002;111:843-50.
5. Lee V, Stoffel M. Bone Marrow: an extra-pancreatic hideout for
the elusive pancreatic stem cell? Journal of Clinical Investment.
2003;111:799-801.
6. Meyer J. Human embryonic stem cells and respect for life.
Journal of Medical Ethics. 2000;26:166-70.
7. Soria B, et al. Insulin secreting cells derived from emdryonic
stem cells normalize glycemia in streptozotocoin induced
diabetic mice. Diabetes. 2000;49:157-62.
8. US Department of Health and Human Services. Stem Cells:
Scientific Progress and Future Research Directions. 2001.
<http://www.nih.gov/news/stemcell/scireport htm>
9. Voltarelli TC, et al. Stem cell therapies for type 1 diabetes
mellitus. Indian Journal of Experimental biology. 2011;49:395-
400.
10. Yang L, et al. In vitro trans-differentiation of adult hepatic stem
cells into pancreatic endocrine hormone-producing cells.
Proceedings of the National Academy of Sciences. USA. 2002;
99:8078-83.
Stem Cell Therapy in Peripheral
Artery Disease
Bimal Kumar Agrawal, Mini Bhatnagar Sud
Introduction
Peripheral artery disease (PAD) refers to atherosclerotic
occlusive disease afflicting arteries other than coronary
arteries; most commonly of the lower limbs but also includes
renal and mesenteric vessels as well as abdominal aorta. As
per statistics available from America the prevalence of PAD is
12–20% in the age group >60 years. The disease is associated
with significant morbidity and mortality with almost half
the patients dying within 4–5 years. The risk factors of PAD
predictably include Diabetes mellitus, hypercholesterolemia,
hyperhomocysteinemia , hyperfibrinogenemia, elevated CRP
and Cystatin C (raised Cystatin and chronic kidney disease is
a significant risk factor for PAD). This article shall highlight
the application of stem cell technology in the treatment of
peripheral artery disease affecting the lower limb vessels.
Risk Profile of PAD
• Age <50 years and presence of other risk factors like diabe-
tes, dyslipidemia, hypertension and raised homocystine.
• Age 50–69 years and history of smoking or diabetes.
• Age 70 or older.
• Leg symptoms with exercise suggestive of claudication.
• Abnormal lower extremity pulse examination.
• Known atherosclerotic coronary, carotid or renal artery
disease.
A vascular examination must be done in any person with
exertional limitation of lower extremity muscles or walking
impairment, i.e. fatigue and aching, poorly healing wounds
or nonhealing wounds of legs or feet, rest pain, post prandial
abdominal pain reproducible on eating and associated with
weight loss and a first degree relative with abdominal aortic
aneurysm.
A blood pressure difference of more than 15 mm Hg
between the two arms, presence of inter arm asymmetry,
bruits, increased diameter of aortic pulsation and absence
of limb pulses is suggestive of PAD. In the distal extremity,
coolness, loss of hair, trophic skin changes of skin and nail
54
changes suggest PAD. Pulse intensity may be graded as
0-absent, 1-diminished, 2-normal and 3-bounding. CLI or
critical limb ischemia means limb pain at rest or impending
limb loss caused by severe compromise of blood flow of the
extremity.
Pathogenesis of PAD includes reduced nitric oxide
levels and activity, free radical damage, proinflammatory
cytokines and increased propensity of thrombosis resulting
in accelerated endothelial damage.1 The global increase in
Diabetes cases and smoking habit has resulted in increasing
detection of this condition.
Investigations for PAD include ankle brachial index (ABI)
or the difference of blood pressure between ankle and arm
using a hand held Doppler. Normal ABI is 1–1.4 and ABI
<0.9 confirms PAD in persons of age 50–70 years, diabetes,
smoking or other risk factors.
ABI and Diagnosis of PAD
ABI >1.4 (high): Suggests a non compressible calcified
vessel. In diabetics and elderly persons even in absence of
arterial occlusion the limb vessels may be fibrotic or calcified
thus resistant to collapse by the blood pressure cuff and a
signal may be heard at higher cuff pressures in these cases.
Here pulse volume recording or toe brachial index by duplex
ultrasound may be done.
ABI = 1-1.4 (borderline to normal): Post exercise ABI may be
done in case symptoms suggestive of claudication are present.
ABI <0.9-(abnormal): Confirmed PAD. Sensitivity of 90%
and specificity of 98% for detecting arterial stenosis of more
than 50% of the lumen. ABI< 0.5 indicates severe stenosis.
For clinically predisposed asymptomatic patients ABI can
be used as a diagnostic test.
Symptomatic patients with claudication symptoms may
be subjected to ABI and pulse volume recording (PVR) by
duplex ultrasound.
Possible claudication may be confirmed by post exercise
ABI.
194  Section 2  Cellular Therapies
Risk factor modification is a very important part of
treatment of PAD.
Smoking cessation, treatment of hypertension and
hyperlipidemia and diabetes, pharmacological risk reduction
using antiplatelet agents Aspirin and clopidogrel can be
used for occluded vessels as well as for cardiovascular
risk reduction. In addition Cilostazol has shown some
improvement in walking time in the absence of heart failure.
Pentoxifylline can be used as second line agent. Others like
iloprost, vitamin E, L-arginine, propionyl L-carnitine and
gingko biloba have not been shown to be of benefit.
Patients should be advised to join a therapeutic
claudication exercise programme.
Revascularization may be attempted by thrombolysis,
endovascular interventions or surgery and if the limb cannot
be salvaged, amputation.
Though most of the cases of PAD are of atherosclerotic
in origin, a small subset of patients has Burgers disease
(Thromboangitis obliterans). As the name implies there is
inflammation of the vessels. Its main etiological association
has been smoking.
Peripheral Arterial Disease: Salient Points
• Atherosclerotic (mostly) in origin, the prevalence increases
with age.
• Risk factors: Hypertension, hyperglycemia, hyperlipi-
demia, smoking, chronic kidney disease.
• Clinical features: Most patients are asymptomatic in early
stage.
– Intermittent claudication
– Thinning of muscles, hair loss, and smooth shiny skin,
reduced or absent pulses.
Critical limb ischemia (CLI), sometime referred to as
end stage PAD-rest pain, ischemic ulcers, risk of loss of
limb significantly increases.
• Diagnosis: It is easy provided it is clinically suspected. ABI
is a simple noninvasive test.
• Treatment: Awareness and prevention of the disease is
ideal. Modification of risk factors and exercise are very
useful if implemented early.
– Pharmacotherapy like cilostazole and pentoxyfylline
has only a modest effect.
– Revascularization may help in some patients, however,
in many it is not feasible, and amputation becomes
necessary in some of them, it is at this stage probably
that the stem cell therapy may play a significant role.
Stem cell technology has evoked interest due to its
potential therapeutic benefit in a wide range of medical and
surgical conditions especially in the field of regenerative
medicine. Stem cells are primordial cells common to multi
cellular organisms. They retain the ability to renew them and
can differentiate into specialized cell types forming various
tissues in early life and growth. They constitute the body’s
internal repair system and replace damaged cells. Stem cells
are distinguished from other cells by two characteristics.
1) Stem cells are unspecialized cells that can renew
themselves through cell division sometimes after long periods
of dormancy, 2) Under certain physiological or experimental
conditions they can be induced to become tissue or organ
specific cells with specialized function. In the gut or bone
marrow stem cells regularly divide to replace damaged tissue
but in the heart and pancreas they divide only under special
conditions.
Stem cells are of two types Embryonic and non embryonic
including somatic or adult stem cells like Mesenchymal stem
cells (MSC), adipose tissue stem cells, neural stem cells,
olfactory stem cells, bone marrow Multipotent stem cells
(MPC) and endothelial progenitor cells (PC). Under certain
conditions specialized adult cells can be reprogrammed
genetically to assume a stem-like state. These are called
induced pluripotent stem cells (i PSC) and have also
generated interest in medical circles.
Due to their unique regenerative abilities stem cells offer
new potential for the treatment of a vast variety of medical
conditions using cell based therapies. This upcoming field is
called Reparative or Regenerative medicine.
Stem cell therapy in the current scenario can be of the
following types:
1. Allogenic stem cell therapy—matched or unmatched
2. Syngenic stem cell therapy—using identical twin
3. Autologous stem cell therapy
4. Cord blood based
5. Nonmyelooablative (lesser intensity transplant or mini
transplant).
Stem cell therapy has been the focus of clinical trials
involving myriad range of conditions including cancer,
neurological degenerative, vascular and traumatic diseases,
autoimmune disease, wound healing, ophthalmological, and
hepatic and bone pathologies. In the present scenario the
complications of stem cell based treatment which limit its
use are infection, regimen toxicity, carcinogenic potential,
immune deficiency and ethical issues surrounding the use of
human embryos.
Stem cell therapy for PAD
Stem cell technology offers new possibilities for angiogenesis
in peripheral artery disease. Stem cells may hypothetically
improve outcome by providing growth factors necessary for
vascular regeneration and providing structural framework
for vascular regeneration. Angiogenesis and arteriogenesis
are two natural compensating mechanisms that occur
with occlusion of peripheral arteries. Angiogenesis refers to
capillary growth that occurs due to release of chemokines and
cytokines like vascular endothelial growth factors (VEGF). The
release of these cytokines is triggered by hypoxia. Sprouting
of small endothelial tubes from pre-existing capillary beds
 Chapter 54  Stem Cell Therapy in Peripheral Artery Disease  195
are not enough to compensate for a large occluded vessel.
Arteriogenesis refers to growth of small collateral branches
from sides of arterial wall. Intra-arterial pressure build-up
within the vessel proximal to the occlusion increases the
shearing forces against vessel wall which induces increment
of diameter of pre-existing collateral arterioles.
It has been proposed that bone marrow derived
mononuclear cells are central to such regenerative and
reparative processes of angiogenesis and arteriogenesis.
These BMMNCs (stem cells) adhere to and invade the vessel
wall. It is not clear whether these invaded cells proliferate
and differentiate into endothelial cells. A growing number of
studies suggest that these circulating EPCs/BMMNCs do not
engraft into blood vessel, rather they regulate vascular repair
via paracrine mechanism by release of cytokines which result
in proliferation of resident endothelial cells. Probably these
BMMNCs are circulating endothelial progenitor cells. It has
been observed that patients with risk factors like diabetes,
smoking, advanced age have lower number of circulating
BMMNCs.
Stem cell therapy in PAD is intended to boost the natural
reparative process of angio- and arteriogenesis. But it requires
large number of functional autologous precursor cells.
About 250–500 mL of bone marrow aspirated and centrifuged
to prepare Buffy coat concentrate. Another method is to
collect these myelo-monocytoid cells (CD34+/FLK1+) from
peripheral blood following injections of GM-CSF. Stem cells
after being isolated is either injected intra-arterially or into
the gastrocnemius (multiple sites, 20 to 30) muscle.
Measures of Outcome of Stem Cell Therapy
Amputation free survival (AFS)—composite end point of
mortality and amputation.
Improvement in Rutherford stage of limb ischemia due
to PAD—category 0-Asymptomatic, category 1-mild claudi-
cation, category 2-moderate claudication, category 3-severe
claudication, category 4-rest pain, category 5-ischemic ulcer-
ation of digits, category 6-extensive ulceration or gangrene.
• Ulcer healing
• Pain free walking (400 meter walk)
• Transcutaneous oxygen pressure (TrO2)-measured by
oxymonitor, normal value is 70–90 mm Hg
• Significant improvement in ABI (increase in ABI =/> 0.1).
Clinical Trials on stem cell therapy
for PAD
Arai et al.3 divided 39 patients of atherosclerotic PAD into
3 groups subjecting the first group to conventional drug
therapy alone, the second to conventional drugs and BMT
while the third received conventional drugs and G-CSF
injections. After one month it was noted that there was
improvement in symptoms in the G-CSF and BMT groups.
ABI and Transcutaneous oxygen pressure (trCut O2) also
improved in the same groups.
Idei et al.4 studying the long term clinical outcome of CLI
patients showed that in 25 patients who received BMMNCs
the 4 year AFS WAS 48% as compared to 0% in PAD controls
who did not get stem cells. The 4 year survival rate was 76% in
the BMMNCs group and 67% in the control group.
Balber5 suggested aldehyde dehydrogenase rich bright
cells (ALDHbr) could be used for angiogenesis in CLI
patients.
Perin et al.6 compared 2 groups of patients of CLI one
of whom received ALDHbr with the other that received
autologous Bone Marrow derived mononuclear cells
(ABMMNCs) and concluded that stastically significant
improvement was observed in Rutherford category at 12
weeks (mean, 4.09+/–0.03 to 3.46+/–1.04; p==0.05) and in ABI
at 6 and 12 weeks.
Huang P et al.7 randomized 28 patients of diabetes
with critical limb ischemia and studied the effect of G-CSF
mobilized PMNCs administered by intramuscular injection
of affected limb. Mean ABI increased from 0.50+/–0.21 to
0.6+/–0.25 (p<0.001). Out of 18 foot ulcers 14 healed in the
transplanted patients and no amputations were required
whereas in the control group 5 amputations were performed
(p=0.007 transplant vs Control).
Tateishi-Yuyama8 et al. studied 25 patients of unilateral
limb ischemia in a randomized controlled trial by injecting
BMMNCs including endothelial PCs into gastrocnemius
muscle of ischemic limb and saline in the less ischemic limb
and in the second group they took patients of bilateral limb
ischemia and randomly injected one limb with BMMNCs
and the other with PMNCs as control. In the transplant group
there was improvement in ABI in the limb with BMMNCs as
compared to that with PMNCs diff 0.009 (95% CI 0.06–0.11;
p<0.0001, transcutaneous oxygen pressure 13(9-17); p<0.001
and pain free walking time (1.2(0.7-1.7)); p=0.025.
Iafrati MD9 et al. in a multicenter double-blind randomized
control trial involving 48 patients with no option CLI who
received bone marrow concentrate injected percutaneuos
in the ischemic limb and controls who received dilute blood
injections showed statistically insignificant trends favoring
the bone marrow recipients vs control in major amputation
(17.6% vs 28.6%), improved pain (44% vs 25%) improved ABI
(32.4% vs 7.1%).
Watt and Gullo10 were of the opinion that MSCs have
tremendous potential as they are expandable ex vivo and
as such may be used to re-establish vascular supply by
regeneration new blood vessels in ischemic tissues. However,
concerns remain about their ability to differentiate into
vascular cells or cardiomyocytes. Their efficacy, safety,
delivery route and dose also need to be standardized by
further clinical trials.
Wang et al.11 conducted a meta-analysis sourced from
PubMed, Cochrane Central Registry of controlled trials,
Elsevier database and EBSCO for autologous cell therapy up
to October 30 2013 and found that clinical end points, i.e.
ABI, Tr-cutO2 pressure, pain scale and AFS were improved
following autologous stem cell therapy in patients with CLI
196  Section 2  Cellular Therapies
but the limited duration of follow-up and limited number of
randomized controlled trials limited their importance.
Current status of stem cell therapy
in PAD
Most trials testing the efficacy and safety of stem cell therapy
in PAD involved a small number of patients who were
suffering from critical limb ischemia but were not candidates
for percutaneuos or surgical therapeutic intervention. There
were few randomized, double blinded multicenter placebo
controlled trials.1,2,11
Trials have shown that endothelial progenitor cells are
vasculogenic as they provide structural framework and
growth factors for vascular regeneration.2 Pericytes also
provide structural framework and may be used. Lack of
knowledge of the exact mechanism of action has limited our
selection of best type of cells to be used but most studies in
the field of vascular regeneration have used ABMMNCs,
Endothelial progenitor cells, Autologous CD34+ Cells or
Aldehyde dehydrogenase rich bright cells (ALDHbr) collected
with the help of flow cytometry from autologous bone
marrow or G-CSF mobilized peripheral blood cells.5,6 The
route of administration used in these trials was intramuscular
injections in the ischemic limb, intrarterial or percutaneous.
The dose of stem cell and duration of follow-up was variable.
Clinical end points of most trials included Ankle brachial
index, transcutaneous oxygen tension (TcO2), duration of
pain free walking and amputation free survival (AFS). Most
trials concluded that stem cell therapy resulted in significant
improvement in ABI, duration of pain free walking and AFS.
Summary
A new era of medical science has begun with the advent of
stem cell technology in the field of therapeutics. Clinical trials
of this treatment modality in the field of peripheral artery
disease have shown promising results in limb salvage and
symptom relief. Though limited by small sample size, lack of
standardized procedures and paucity of placebo controlled,
randomized, double blinded multicenter studies; yet it
may be hoped that stem cell therapy will find a place in the
treatment of PAD in future when more refined techniques
shall be developed and tested and proven to be effective.
Glossary of terms
ABMT—Autologous bone marrow transplant.
ABI—Ankle brachial index.
AFS—Amputation free survival.
ALDHbr—Aldehyde dehydrogenase rich bright cells, hemat-
opoietic stem cells are rich in aldehyde dehydrogenase. To
isolate stem cells a fluorescent substrate is used which makes
the cell bright.
BMMNCs—Bone marrow derived mono nuclear cells.
CLI—Critical limb ischemia.
G-CSF—Granulocyte colony stimulating growth factor.
iPSCs—Induced pluripotent stem cells, genes expressed in
embryonic stem cell is introduced into adult cells (mature
differentiated cells), which are then dedifferentiated into
pluripotent stem cells.
MSCs—Mesenchymal stem cells, sometime called as marrow
stromal cells-these are multipotent cell that can differentiate
into a variety of cells like osteoblasts, chondrocytes,
adipocytes.
PSCs—Progenitor stem cells, early descendants of stem cells
that can differentiate into one or more kind of cells but cannot
divide and reproduce indefinitely.
PMNCs—Peripheral blood mono nuclear stem cells.
References
1. Crystal M Botham, William L, et al. Clinical trials of adult stem
cell therapy for peripheral artery disease. MDCVJ/IX 2013.
2. Thekkeparambil Srijaya, Thamil Ramasamy, Noor Kasim.
Advancing stem cell therapy from bench to bedside; lessons
from pharmaceutics for bio-pharmacy. Journal of Translational
Medicine. 2014;12:243.
3. Arai M, Misao Y, Nagai H, Kawasaki M, Nagasimha K, Suzuki
K, et al. Granulocyte-colony stimulating factor. A non-invasive
therapy for treating atherosclerotic peripheral artery disease.
Circ J. 2006;70(9):1093-8.
4. Idei N, Soqua J, Hota T, et al. Autologous bone marrow
mononuclear cell implantation reduces long-term amputation
risk in patients with critical limb ischemia. A comparison of
atherosclerotic peripheral artery disease and Burgers’ disease.
Circ. Cardiovascular Interv. 2011;4(1):15-28.
5. Balber AE. Concise review; aldehyde dehydrogenase bright
stem cells and progenitor cell population from normal tissue.
Characteristics activities and emerging uses in regenerative
medicine.
6. Perin EC, Silva G, Grahremanpour A, et al. A randomised
controlled study of autologous therapy bone marrow derived
aldehyde dehydrogenase bright cells in patients with CLI.
Catheter Cardiovascular Interv. 2011;78(7):1060-7.
7. Huang P, Li S, Han M, Xiao Z, Yang R, Han ZC. Autologous
transplantation of Granulocyte colony stimulating factor
mobilised peripheral blood mononuclear cells improves critical
limb ischemia in diabetes. Diabetes Care. 2005;28(9):2155-60.
8. Tateishi-Yuyama E, Matsubara H, MuroharaT, Ikeda U, Shintai
S, Masaki H, et al. Therapeutic angiogenesis for patients with
limb ischemia by autologous transplantation of bone marrow
cells: A pilot study and a randomised control trial. Lancet.
2002;360(9331):427-35.
9. Iafrati MD, Hallett JW, Geils G, Pearl G, Lumsden A, Peden E,
et al. Early and lessons learnt from a multicentre, randomised,
double-blind trial of bone marrow aspirate concentrate in
critical limb ischemia. J Vasc Surg. 2011;1:54(6):1650-8.
10. Watt SM, Gullo. Angigenic properties of Mesenchymal stem/
stromal cells and their therapeutic potential. Br Med Bull.
2013;108:25-33.
11. Wang ZX, Li D, Cao JX, et al. Efficacy of autologous bone
marrow mono nuclear cells therapy in patients with peripheral
artery disease. J Atherosclerosis Thromb. 2014.
Role of Platelet Rich Plasma
in Regenerative Medicine
Kusum K Thakur, Alpna Thakur, Achhar Singh
INTRODUCTION
Regenerative medicine is one of the new therapeutic
approaches characterized by biological regeneration of the
cells and tissues instead of their replacement.1 Thus, it is an
exciting new field of medicine that seeks to repair, rebuild and
restore damaged tissue by maximizing the use of the body’s
own natural healing processes. Our body has an amazing
capacity to heal itself, but sometimes this process falls short
or remains incomplete. Regenerative medicine seeks to help
the body heal itself by concentrating and delivering powerful
healing agents like platelets and stem cells directly to the
site of tissue injury. This restarts and maximizes the healing
process in a natural way.2 Platelet rich plasma (PRP) therapy
has been used extensively in Europe for several years and is
now becoming more popular in the United States as more
people become aware of its potential benefits and as more
research is being done.3 In India, recently a study by PK
Sehgal et al. showed decrease in wound surface area which
was statistically significant. The mean healed surface area
of the pressure ulcers reduced at the final follow-up. After
5 weeks of PRP therapy, 60% of the wounds had shown well-
formed granulation with epithelization on histopathology.
In this study, 96% of the pressure ulcers improved after PRP
therapy.4
HISTORICAL PERSPECTIVE
Prolotherapy was “discovered” in the 1930s by Dr Earl
Gedney, an osteopathic surgeon, before the term regenera­
tive medicine existed. However, prolotherapy is a true
regenerative medicine, working by locally raising growth
factor levels to promote tissue repair and regeneration. In
the 2000s, in office platelet rich plasma (PRP) prolotherapy
was introduced. The newest from of prolotherapy, known
as stem cell prolotherapy, is used in difficult cases or where
accelerated musculoskeletal healing is desired.5
Some clinical applications in which PRP was used to treat
and repair injured tissues and vessels in cutaneous ulcers
55
have been carried out since 1980s. Later in 1990s, platelets
were introduced into maxillofacial surgery as fibrin glues.
The clinical potential of PRP-therapies has truly achieved
positive clinical results in enhanced bone formation and
anti-inflammatory functions during oral and maxillofacial
applications since that time. To date, PRP has been used
in the treatment of more than 30 diseases that belonged to
facial rejuvenation and plastic surgery, maxillofacial surgery,
dentistry and oral surgery, tissue engineering and research,
cardiovascular surgery, orthopedic surgery, and sports
medicine, gastroenterology and urology6 as shown in Figure 1.
Figure 1  Timeline for platelet rich plasma applications
BASIC SCIENCE OF PLATELET RICH PLASMA
Platelets are small, non-nucleated bodies in peripheral blood
that are known primarily for their role in hemostasis.6 These
contain numerous granules, which are reservoirs of growth
factors and various cytokines needed for cell repair and
regeneration.
Platelet rich plasma (PRP), with a concentration above
baseline is used in PRP therapy. In fact, PRP must have a
minimum increase of five times the normal concentration of
platelets (1000 × 109/L).7 It is one of autologous product which
enhances the recruitment, proliferation, and differentiation
of cells involved in tissue regeneration. Platelets contain a
198  Section 2  Cellular Therapies

Table 1: Growth factors identified within platelet rich plasma and their physiologic effect
Factor Target cell/ tissue Function
PD-EGF Blood vessel cells, outer skin cells Cell growth, recruitment, differentiation, skin closure cytokine
fibroblasts and many other cell types secretion

PDGF A+B Fibroblasts, smooth muscle cells, chondrocytes, Potent cell growth, recruitment, blood vessel growth, granulation,
osteoblasts, mesenchymal stem cells growth factor secretion; matrix formation with BMPs (collagen and
bone)

TGF-β1 Blood vessel tissue, outer skin cells Blood vessel (+), collagen synthesis
fibroblasts, monocytes TGF gene family includes the growth inhibition, apoptosis (cell death)
BMPs, osteoblasts-highest levels of TGF-Br differentiation, activation
IFG-I, II Bone, blood vessel, skin, other tissues Cell growth, differentiation, recruitment collagen synthesis with
fibroblasts PDGF
VEFG, ECGF Blood vessel cells Cell growth, migration, new blood vessel growth, anti-apoptosis
(anti-cell death)
bFGF Blood vessels, smooth muscle, skin Cell growth, cell migration, blood vessel growth
fibroblasts, other cell types
Abbreviations: PD-EGF, platelet-derived epidermal growth factor; PDGF, platelet-derived growth factor; BMP, bone morphogenetic protein; TFG, transforming
growth factor; IGF, insulin-like growth factor; VEGF, vascular endothelial growth factor; ECGF, endothelial cell growth factor; bFGF, basic fibroblast growth factor.

number of proteins, cytokines and other bioactive factor as
shown in Table 1 that initiate and regulate basic aspects of
wound healing most notably in acute surgical conditions and
in the management of chronic nonhealing wounds.6
PLATELET RICH PLASMA THERAPY
This therapy is safe and natural alternative to surgery. Platelets
contain an abundance of growth factors and cytokines that
can affect inflammation, postoperative blood loss, infection,
osteogenesis, wound, muscle tear and soft tissue healing.
They also release many bioactive proteins responsible
for attracting macrophages, mesenchymal stem cells and
osteoblasts that not only promote removal of degenerated
and necrotic tissue, but also enhance tissue regeneration and
healing. This therapy relieves pain by promoting long lasting
healing. PRP therapy relieves pain without the risk of surgery,
general anesthesia or hospital stays and without a prolonged
recovery. In fact most people return to their jobs right after
the procedure.3
(30–40 mL) from patient‘s elbow vein with a syringe to a
test tube which is anti-coagulated with citrate phosphate
dextrose adenine (CPDA) with a ratio of 1 : 9 to the blood, to
bind ionized calcium and inhibit the clotting cascade.
It is followed by two centrifugations, one ten minutes
centrifugation at 2000 rpm which separates blood into three
layers; red blood cells, platelets and platelet poor plasma as
shown in Figure 2.
Red cell layer is drawn from the tube. The remainder
is agitated for several seconds and will undergo a second
centrifugation at 2000 rpm for ten minutes. There will be two
layers, upper supernatant is platelet poor plasma (PPP) and
residual is platelet concentrates. About the three fourth of

PREPARATION OF PLATELET RICH PLASMA

AND ITS APPLICATION
Blood consists of approximately 93% of red blood cells
(RBCs), 01% white blood cells (WBCs) and 06% of platelets,
all suspended in plasma. PRP can only be made from
anticoagulated blood.7 Preparation of PRP is done in
the department of transfusion medicine using standard
preparation technique on the day of application from the
patient’s own blood. It begins by a small amount of blood Figure 2  Layers of blood after first centrifugation
 Chapter 55  Role of Platelet Rich Plasma in Regenerative Medicine  199
supernatant is discarded and residual platelet concentrate
is drawn into a syringe. Another syringe with 10% calcium
chloride will be integrated with PRP syringe in a ratio of 6 : 1 to
activate the PRP for delivery to the desired site.4 Clotting leads
to platelet activation, resulting in release of growth factors
from alpha granules. Approximately 70% of stored growth
factors are released within 10 minutes of PRP activation, and
nearly 100% within one hour and rest till 8–10 days.6 PRP is
injected/applied to and around the point of injury/wound.
The procedure time takes about one to two hours, including
preparation and recovery time. Twice weekly dressings
done for maximum ten dressings for chronic wounds and
maximum up to three injections can be given for injuries
within six months time frame, usually performed two to three
weeks apart but considerable or complete relief may be gained
after first to second injection.3,4 Before application informed
patient’s consent is must after explaining the procedure, risk
and benefits.4
THERAPEUTIC PLATELET PRODUCTS8
• Platelet rich plasma: Platelets concentrate obtained after
two centrifugations as described above.
• Platelet gel: Platelets are exposed to an agonist, which
induces activation resulting in release of intrinsic
substances that are applied to the target area where they
accelerate wound healing.
• Platelet leukocyte gel: Contain high concentrations of
platelets and leucocytes with antimicrobial properties in
addition, at wound.
INDICATIONS
• Dermatology and esthetic surgery: Fine wrinkles around
the eyes, bags and dark circles under the eyes, neck, chest
and décolletage, lips, around the ears, cheeks, naso-
labial folds, acne and scars, back of hands and arms, hair
growth and change of color. It is safe and effective tool
in the management of mucosal skin lesions related to
the graft-versus-host disease (GVHD) involving dermis
subcutaneous or oral mucosa and is applied as local
therapy (Figs 3A and B).8
• Orthopedics: Tendonitis, ligament sprains, intra-
articular injuries, joint pain, arthritis, knee meniscus
repair, acute and/or chronic muscle strain. PRP therapy
is a revolutionary new treatment that relieves pain by
promoting lasting healing of musculoskeletal injuries
(Figs 3G to I).3,8
• Oral and craniofacial surgeries: Maxillofacial settings
where it is considered standard treatment of bone defects,
artificial endosseal implants, oral reconstructions,
maxillary sinus augmentation (Fig. 3F).8
• Ophthalmology: PRP has been successfully used for ocular
diseases, such as for dry eye syndrome and enhanced
healing of corneal surface and ocular surgery (Fig. 3L).8,9
• Chronic wound healing: In the United States, the
diabetic foot (Fig. 3J) problem accounts for 20% of all
diabetic hospital admissions and 50% of all nontraumatic
amputations. Ulcers occur in 15% of diabetics, and 6–20%
of all hospitalized diabetic patients have foot ulcers.8,10
The topical use of the platelet rich plasma may play an
important role in the initiation of the repair process
of chronic wounds (Fig. 3E). Various protocols have
documented such an improved healing of surgical wound
in cardiac surgery (Figs 3C and D) and plastic surgery
(Fig. 3K).
• Cancer: PRP has a definite role in the cancer, which is
probably due to their proangiogenesis stimulation and
regulation of new blood vessel growth through numerous
stimulators and inhibitors of angiogenesis, such as
endostatin and VEGF, by several pathways.8
• Neurology: There is role of PRP therapy in cases of
ischemic damage to a nerve due to scar tissue banding
during percutaneous procedures.8
• Other applications: Platelet gel mixed with centrifuged
fat has been successfully applied for the patients affected
by Parry-Romberg syndrome, i.e. progressive hemifacial
atrophy and volumetric deficit, which is an uncommon
degenerative and poorly understood condition.8
CONTRAINDICATIONS11
• Absolute: Platelet dysfunction syndrome, critical
thrombocytopenia, hemodynamic instability, septicemia,
local infection at the site of procedure and patient
unwilling to accept risks.
• Relative: Consistent use of NSAIDS within 48 hours of
procedure, corticosteroid injection at treatment site
within one month, systemic use of corticosteroids within
two weeks, tobacco use, recent fever or illness, cancer
hematopoietic or bone, hemoglobin less than 10 g/dL and
platelet count less than 100 × 109/L.7
SAFETY PROFILE
Since PRP is prepared from autologous blood, there are
minimal risks for disease transmission, immunogenic
reaction or cancer as compared to standard therapies. Till
date, PRP has been widely used in the treatment of at least
thirty diseases in more than hundred thousand patients,
and there have been no reports about side effects of PRP
injection.5
SUMMARY
Studies have shown that with PRP application, there
is complete absence of keloid formation, hyperplasia,
metaplasia or dyschromia of healed lesion, comfort and pain
diminution, less edema and ecchymosis, reduced mortality
after cardiac surgery, reduced infection rate after cardiac and
200  Section 2  Cellular Therapies
A D
B E
C F
Figures 3A to L:  Pictures showing some of the applications of
surgery; E: Chronic wound healing, F: Dentistry; G to I: Orthopedics and musculoskeletal pain relief; J: Diabetic wound healing, K: Plastic surgery;
L: Cosmetic ophthalmic surgery
orthopedic surgery, reduced transfusion supply and hospital
stay after orthopedic surgery and reduced amputation rate
in diabetic foot. Practically there are no or minimal ill effects
when compared to standard therapies.8
FUTURE OF PLATELET RICH PLASMA
IN REGENERATIVE MEDICINE
Based on present scenario of PRP application, it will widely
be applied in almost all wounds as well as chronic diseases
in combination with stem cells. In the future, a standard
operating procedure in PRP preparation is essential. The
artificial PRP should also be suggested for use in future.7
CONCLUSION
Platelet rich plasma is a natural biological product which
can be used as a drug in wound treatment, as an adjuvant
in stem cell transplantation, as a differentiating factor in
differentiation of stem cells and stimulator in stem cell
culture.7 Many in vitro studies have established that platelet-
derived growth factors accelerate proliferation of an array
of cells involved in soft and bony tissue regeneration.
These effects have also been evaluated and confirmed by
numerous in vivo, both in animal and human clinical studies.
G J
H K
I L
PRP12-23:A and B : Dermatology and esthetic use; C and D : Cardiothoracic
Consequently, we can say that the platelet-derived products
currently represent a valuable therapeutic modality, offering
opportunities for various applications in regenerative
medicine and tissue engineering.8
REFERENCES
1. Sabin e Zenker: Controversies in regenerative medicine,
IMCAS Annual Meeting, Paris, 2010. pp. 8-11.
2. Tellis AA. Crystal coast management center, 2111 Neuse Blvd.
Suite J, New Bern, North California. Regenerative Medicine:
Using Prp And Stem Cells As A Natural, Powerful Alternative
for the Management of Chronic Pain.
3. http://www.treatingpain.com-platelet rich plasma therapy.
4. Sehgal PK, et al. At Pt. BD Sharma PGIMS, Rohtak, Haryana
2013. “Role of local application of autologous platelet-rich
plasma in the management of pressure ulcers” presented
at 38th ISBTI National Conference at Surat and 2nd ISTM
Conference at Banglore.
5. Alderman DD, Alexander RW. Advances in Regenerative
Medicine: High-density Platelet-rich Plasma and Stem
Cell Prolotherapy for Musculoskeletal Pain, Practical Pain
Management. 2011.pp.22-25.
6. Hara G, Basu T. Platelet-rich Plasma in regenerative medicine.
Biomedical Research and Therapy. 2014;1(1):25-31.
7. Foster TE, et al. Platelet Rich Plasma (PRP); from basic
sciences to clinical application. The American Journal of Sports
Medicine. 2009.pp.1-10.
 Chapter 55  Role of Platelet Rich Plasma in Regenerative Medicine  201
8. Rozman P, et al. The Role of Platelet Gel in Regenerative
Medicine, Advances in Regenerative Medicine, Dr. Sabine
Wislet-Gendebien (Ed.), 2011. ISBN: 978-953-307-732-1.
9. Freire Y, et al. in vitro effects of PRP on human corneal epithelial
cells. 2012;53:5571-8.
10. Margolis DJ, Kantor J, Santanna J, et al. Effectiveness of platelet
releasate for the treatment of diabetic neuropathic foot ulcers.
Diabetes Care. 2001;24:483-8.
11. Guidelines for use of PRP by The International Cellular Medical
Society: DRAFT Version 1.0.
12. http://www.miamidermatologycenter.com/platelet-rich-
plasma-facelift.
13. http://mauiregenerativemedicine.wordpress.com.
14. http://www.drlox.com/Can-Platelet-Rich-Plasma-be-used-
for-all-Joints.
15. http://www.intechopen.com/books/skin-grafts-indications-
applications-and-current-research/recent-innovation-in-
pretreatment-for-skin-grafts-using-regenerative-medicine-in-
the-east.
16. http://r3stemcell.com/2014/03/17/faqs-regenerative-
medicine.
17. http://www.woundsresearch.com/files/wounds/yuan2.png.
18. http://www.cosmosclinic.com.au/face/prp-platelet-rich-
plasma/.
19. http://www.cosmeticimageclinics.com.au/cosmetic-surgery-
before-and-after-photos/.
20. https://www.dentalaegis.com/id/2013/05/a-clinical-therapy-
for-the-treatment-of-peri-implantitis.
21. http://www.terumo-ccvs.com/optimizing/southeast missouri
hospital finds cost advantages using platelet gel.shtml.
22. http://www.whatclinic.com/body-treatments/uk/hampshire/
winchester/evolutions-clinic.
23. http://www.itemcliniceng.com/antiaging/item-antiaging09.
html.
Role of Platelet Rich Plasma
in Plastic Surgery
Umesh Sharma
Platelet Rich Plasma
Platelets are more than just first-line responders to bleeding
injury. Every platelet is also a biochemical storehouse of
regulatory, signaling and growth-factor molecules that
participate in recovery and healing of tissue as well as
emergency response to injury. Growth-factor molecules
associated with platelets include:
• Platelet-derived growth factor (PDGF): Promotes blood
vessel growth, cell replication, skin formation.
• Transforming growth-factor-beta (TGF-b): Promotes
growth of matrix between cells, bone metabolism.
• Vascular endothelial growth factor (VEGF): Promotes
blood vessel formation.
• Epidermal growth factor (EGF): Promotes cell growth
and differentiation, blood vessel formation, collagen
formation.
• Fibroblast growth factor-2 (FGF-2): Promotes growth of
specialized cells and blood vessel formation; and
• Insulin-like growth factor (IGF): A regulator of normal
physiology in nearly every type of cell in the body.
Rationale for use of Platelet
Rich plasma
The rationale for use of PRP in surgery is to artificially increase
the number of platelets at a site where the storehouse of
growth factors in the platelets can be put to use in enhancing
tissue recovery, repair and health.1
Platelet rich plasma (PRP) is also finding increasing use
in orthopedics, dental implant surgery and other surgical
specialties as a wound treatment. Safe use of PRP has been
documented in neurosurgery, ophthalmology, urology,
cardiac surgery, thoracic surgery, maxillofacial surgery and
cosmetic surgery.2,3 Approval of these uses by the US Food
and Drug Administration (FDA) is not required as long as the
use is procedural and part of the treatment process, and no
advertised claims of its efficacy are made.
56
Platelet Rich plasma in Plastic Surgery
Platelet Rich Plasma in Hair Transplantation
The potential for using PRP to promote healing and hair
growth after hair transplantation is centered in three
functional applications:4
1. To preserve and enhance hair follicle viability during and
after hair transplantation.
2. To promote and enhance tissue repair and healing after
hair transplantation; and
3. To reinvigorate dormant hair follicles and stimulate new
hair growth.
To Preserve and Enhance Hair Follicle Viability
Between the time that hair follicles are removed from a donor
area of the scalp and transplanted into a recipient area, they
are subject to damage from several causes:
• Dehydration if the donor follicles are inadequately
moistened between the times of removal and
transplantation.
• Oxygen and nutrient starvation due to being removed
from blood supply during the harvest-to-transplantation
time period.
• Temperature and acid/alkaline changes in the follicle
environment; and
Search for a way to enhance survival of transplanted
hair follicles and promote healing with minimal scarring
after transplantation led to trials of PRP.
A common approach to maintaining donor hair follicle
viability during the transition period is to keep them in a
storage solution that provides a protective environment of
appropriate temperature, chemical balance and nutrient
supply. Recent research has indicated that addition of PRP
to the storage solution improves follicle viability during
and after transplantation, enhances post-transplantation
 Chapter 56  Role of Platelet Rich Plasma in Plastic Surgery  203
tissue healing and promotes hair growth in transplanted
follicles. An approach advocated by some investigators is to
bathe the donor hair follicles in activated PRP just prior to
transplantation.
To Promote and Enhance Tissue Repair
and Healing
In activity promoting tissue repair and healing after injury of
surgery, the growth factors stored in platelets are released at
a site of tissue injury, promoting tissue repair and healing.
Individual growth factors such as PDGF have been used by
surgeons to promote wound healing in hospitalized surgical
patients. The rationale for using PRP in outpatient surgical
hair restoration is to use the full array of platelet-associated
growth factors to promote healing and minimize scar
formation, as well as to promote maximum hair growth in
transplanted follicles.
To Reinvigorate Dormant Hair Follicles
After noting enhanced hair growth of transplanted hair
after use of PRP, investigators conducted a small study of
PRP effect on dormant non-transplanted hair follicles. The
study hypothesized that platelet growth factors can “wake
up” dormant hair follicles and begin the production of new
hair. PRP was applied after scalp skin was slightly injured to
induce platelets to release growth factors at the injury site.
Enhanced hair growth and hair diameter was noted over the
next 4 months, with a fall-off in enhanced hair growth after 4
months. This use of PRP is still regarded as experimental, with
need for further study.5
Skin Rejuvenation
Similarly PRP has been used for facial rejuvenation where
it is used for anti-aging purpose. Here the PRP is injected
intradermally with 30 gauge needle at multiple places
in an attempt to raise a wheal, second injection is given
1–1.5 cm away. The supernatant platelet poor plasma (PPP)
is massaged over the needle prick sites at the completion
of procedure. The procedure is done once a month for 3 to
4 sittings followed by once in four to six months depending
upon the need.
Wound Healing
Healing of chronic wounds, trophic ulcers, sinuses can be
improved to a greater extent by injecting PRP along the
margin of the ulcer and smearing the PRP/Coagulam over
the base of non-healing ulcer. The process leads to rapid
granulation tissue formation with subsequent faster healing;
most of infections are also prevented.
Other uses in plastic surgery are for better adherance
and takeup of skin grafts, injections in scar tissues and
hypertrophic scar to improve their appearance.
Safety, Complications,
Contraindications to Use of
Platelet Rich plasma
Platelet rich plasma (PRP) is immunologically neutral and
poses no danger of allergic, hypersensitivity or foreign-body
reactions. Sterile technique must be used at every stage of PRP
preparation and application. Sterile technique is especially
important if a patient has an underlying medical condition
that predisposes to infection. A brief period of inflammation at
wound sites may be experienced by a patient after application
of a PRP gel. Inflammation may be associated with release of
platelet-associated factors at the wound site.
As use of PRP in medicine is becoming more widespread,
there are also increasing cautionary warnings about uncritical
acceptance of PRP as a “wonder cure”. While the outcome of
PRP treatment is often positive, there is still a lack of unbiased
scientific data from controlled clinical trials in which PRP
treatment is objectively compared with other treatments for
the same condition.
References
1. Arora NS, Ramanayake T, Ren YF, et al. Platelet-rich plasma: a
literature review. Implant Dentistry. 2009;18:303-10.
2. Sampson S, Gerhardt M, Mandelbaum B. Platelet rich plasma
injection grafts for musculoskeletal injuries: a review. Current
Review of Musculoskeletal Medicine. 2008;1(3-4):165-74.
3. Schwartz A. A promising treatment for athletes, in blood.
http://www.nytimes.com/2009/02/17/sports/17blood.html?
4. Cooley J. Hair transplant graft survival and platelet rich plasma.
http://www.regrowhair.com/hair-transplant-surgery/hair-
transplant-graft-survival-and-platelet-rich-plasma.
5. Greco J, Brandt RJ. Maximizing the use of autologous
platelet rich plasma in hair transplantation surgery. http://
grecohairrestoration.com/complex.htm.
Cord Blood Banking and
Transplantation
Paramjit Dhot
Introduction
Stem cells are the mother cells of the human body. Stem cells
are self-renewing and can give rise to more specialized cells
of human body, such as muscle cells, blood cells and brain
cells. They are best described in the context of normal human
development. When a sperm fertilizes an egg, the product is
a single cell that has the potential to form an entire organism.
These stem cells are being used for curing blood cancers
and diseases like thalassemia and cerebral palsy. Clinical
trials are in progress to use the stem cells in heart attack,
diabetes mellitus, spinal injury, osteoarthritis and nonunion
of bones. The sources of stem cells are from the bone marrow,
peripheral blood stem cells, cord blood stem cells and cord
tissue stem cells.
Cord blood transplantation has come of age. Umbilical
cord blood banking efforts have increased dramatically in
the past two decades in response to increasing demands
for alternative sources of stem cells to support patients
requirement stem cell transplantation. Twenty five years after
the first cord blood transplant, approximately 3 million cord
blood units are stored in family cord blood banks all over the
world and 900 cord blood units were released for therapy.1–3
Over 650,000 umbilical cord blood units have been stored
in public banks for transplants worldwide. Over 30,000 cord
blood transplants have been performed globally.3
Cord blood stem cells are collected after the birth of
the baby. After the umbilical cord is clamped and cut,
approximately over 80 mL of cord blood is collected from the
umbilical cord in a blood bag with an anticoagulant. This cord
blood is very rich in stem cells, and after removal of red blood
cells, the stem cells are mixed with a cryoprotectant dimethyl
sulpoxide. These stem cells are stored in liquid nitrogen at
-196°C in liquid nitrogen.4 The shelf life of these stem cells is
21 years.5
These stem cells can be used by the baby, the sibling,
parents and even grandparents after HLA matching.
Placenta and cord blood which was earlier discarded in
the garbage is now providing lifesaving stem cells "Garbage
57
to Gold". The first cord blood transplant was carried out in a
boy with Fanconi’s Anemia by Dr Eliane Gluckman in Paris,
in 1988, from stem cells of his sister and he is well and cured
of his life threatening disease.2
Lifecell International is the first, largest, oldest and most
accredited cord blood bank in the country with over 100
collection centers in India and abroad namely Bahrain,
Dubai, Abu Dhabi and Nepal. More than 100,000 units of cord
blood are stored by Lifecell International at Chennai and dual
storage facility at Manesar. Of these 30 cords blood units have
been retrieved for transplantation. There were 21 cord blood
transplants for Thalassemia Major, 5 for cerebral palsy and
4 for leukemias. The cerebral palsy transplant patients have
shown neurological and functional improvement. A Public
Cord Blood Bank has been launched by Lifecell, on 27 July
2014, to store 10,000 units at a cost of Rs 30 Crores.
The advantages of cord blood are that it is easily available
and has less graft versus host disease. In the coming decade
Cord Blood stem cells will be used extensively in the clinics
and hospitals for various malignant and nonmalignant
disorders.
Cord Blood Transplantation in
Thalassemia in India
Current estimates suggest that approximately 10,000 children
are born each year in India with Thalassemia. Transplantation
of stem cells offers the only hope of cure for a number of
diseases of childhood, both malignant and nonmalignant,
including Thalassemia. The preferred donor is a matching
sibling, but with a growing number of one child families and
only a one in four chance of a sibling being a match, there is an
increasing need for match unrelated donors. Hematopoietic
stem cells transplantation offers the only hope of cure for
a number of diseases of childhood both malignant and
nonmalignant including thalassemia and metabolic storage
disorders.3
Lifecell International, India’s first and most accredited
private Cord Blood Bank offering dual storage facility with
 Chapter 57  Cord Blood Banking and Transplantation  205
over hundred collection centers in India and abroad. Thirty
Cord Blood Transplants have been successfully carried out
till date out of which 21 were for thalassemia. The detailed
patient’s outcome was as follows: the youngest patient was
2 years and oldest 9 years in age. There were 9 males and
12 females. Standard conditioning regimen was used in
all the patients. One patient had lower GI bleed and febrile
neutropenia. Mild hypertension was seen in 5 patients. Major
hypertensive encephalopathy with convulsions due to DMSO
toxicity was seen in 1 patient. One patient had hypotension.
The other transplant patients had a smooth recovery. On
follow up 95% of the patients have shown a disease free
survival.
Ideally the cord blood unit should be 6/6 HLA match with
the patient, but 5/6 or 4/6 are also accepted.6 The required
total nucleated and CD 34 cell dose depends on the recipient
body weight. The disease free survival rate varies between 80
and 95% in beta thalassemia.7
Cord Blood Transplantation in India
The cost of autologous transplant in India is $ 10,000 to
$ 12,000 and allogeneic transplant is $ 18,000 to $ 20,000. The
chance of finding a matching family donor is 25%, whereas
it is one in a million in unrelated donors. In India there is
no bone marrow donor registry and hence patients in India,
face a problem in finding a donor. Approximately 5% of the
transplant patients are lost due to fungal infections in India.
The major transplant centers in India are CMC Vellore, Tata
Memorial Hospital Mumbai, Army Hospital Delhi Cantt,
Tata Medical Centre Kolkata, AIIMS New Delhi and Apollo
Hospitals. Approximately 100 to 125 Umbilical Cord Blood
transplants are carried out in the country annually.
Public Cord Blood Banking
Lifecell International has launched the Public Cord Blood
Programme on 27th July 2014 at Chennai. This will offer HLA
matched cord blood units for the needy patients as a cure for
their ailments.8
With the availability of increasing umbilical cord blood
supply and numerous new indications being added resulting
in markedly improved clinical outcomes in cord blood
transplant patients. It is our hope at Lifecell, that by providing
families in India with a means to store their children’s sibling
cord blood more children will receive cord blood transplants
to cure thalassemia and other inherited disorders.
Conclusion
The absence of ethical concern and the unlimited supply of
cells explain the increasing interest of using cord blood for
regenerative medicine and cellular therapy.
After 26 years of the first cord blood stem cell transplant,
we anticipate an abundant umbilical cord blood supply,
digitalized umbilical cord blood selection, multiple new
indications and significantly improved clinical outcomes.
In India, family cord blood banking was started by lifecell
International in 2004. In the future, it is expected that cord
blood transplantation will be the mainstay in the therapy for
malignant and nonmalignant disorders. Stem cell science
represents to those afflicted with chronic disease a vehicle
for disease free survival. Moreover regenerative medicine,
cellular therapy and tissue engineering will offer a panacea
for the needy patients.
References
1. Ballen KK, Gluckman E, Broxmeyer HE. Umblical cord
blood transplantation: the first 25 years and beyond. Blood.
2013;122:491-8.
2. Gluckman E, Ruggeri A, Rocha V, et al. Family directed umbilical
cord blood banking. Haematologica. 2011;96(11):1701-7.
3. Broxmeyer HE, Farag SS, Rocha V. Cord blood Haematopoietic
Cell Transplantation. In: Appelbaum FR, Forman SJ, Negrin RS,
Antin JH (Eds). Thomas’ Haematopoietic Cell Transplantation.
5th edn. Oxford, England: Wiley Blackwell; 2013.
4. Dhot PS, Nair V, Swarup D, et al. Cord Blood Stem Cell Banking
and Transplantation. Indian J Pediatric. 2003;70(12):989-92.
5. Broxmeyer HE, Lee MR, Hangoc G, et al. Haematopoietic stem/
progenitor cells, generation of induced pluripotent stem cells
and isolation of endothelial progenitors from 21 to 23.5 year
cryopreserved cord blood. Blood. 2011;117(18):4773-7.
6. Eapen M, Klein JP, Ruggeri AL, et al. Impact of allele level
HLA matching on outcomes after myeloablative single unit
umbilical cord blood transplantation for haematologic
malignancy. Blood. 2014;123(1):133-40.
7. Dhot PS. Cord blood transplantation for Thalassaemia in India.
Parent’s Guide to Cord Blood. 2014;6:2-3.
8. Appelbaum FR. Pursuing the goal of a donor for everyone in
need. N Engl J Med. 2012;367(16):1555-6.
Erythropoiesis Stimulating
Agents versus Red Cell Transfusion
Sandeep Puri
INTRODUCTION
Blood transfusion is defined as the transference of blood
from the circulation of one individual to that of another for
practical therapeutic purposes. Although the practical use of
blood transfusion in humans is of relatively recent origin, the
concept of ‘transfusion’ has a longer history. With the discovery
of circulation of blood by William Harvey, there emerged a
new era in medicine. The first documented successful blood
transfusion was done in dogs in 1660’s. Attempts were made
for animal to human transfusions, but it often ended in fatality.
It was only in the early nineteenth century that first human
to human transfusion could be successfully accomplished.
Since then, there has been no looking back. A large number of
previously unknown properties of blood have been unearthed
and the science of transfusion medicine has revolutionized
the modern day therapeutics. Currently, not only whole
blood, but blood components (packed red cells, plasma,
platelets and cryoprecipitate) are too an important medical
armamentarium. However, with the success, also came the
risks of acquiring blood borne infections, immunological
reactions, iron overloads and transfusion related lung injury.
This significantly affected the quality of life of the recipients.
Therefore, there began a search for alternatives to avoid
transfusion.
ERYTHROPOIESIS STIMULATING
AGENTS
In earlier twentieth century, it had been shown that hormones
regulate the production of red cells. Later, it was demonstrated
that a circulating substance stimulated red blood cell
production and increased hematocrit. This substance was
finally purified and confirmed as a hemopoietic agent and
was termed erythropoietin. With demonstration of the gene
for erythropoietin, that is biologically active both in vitro
and in vivo, recombinant human erythropoietin and other
erythropoiesis stimulating agents (ESAs) were synthesized.
ESAs are naturally similar to the protein erythropoietin.1
58
Available erythropoiesis stimulating agents
            Erythropoietin
Short acting ESAs
            Epoetin alfa
            Epoetin beta
            Epoetin zeta
            Epoetin theta
Long acting ESAs
            Darbepoetin alfa
CERA: Continuous erythropoietin receptor activator
In 1989, the US Food and Drug Administration approved the
hormone for clinical use.
Erythropoiesis stimulating agents (ESAs) are approved
primarily for the treatment of anemia resulting from chronic
kidney failure, in patients with cancer chemotherapy induced
anemia, in patients with HIV whose anemia is caused by
Zidovudine and to reduce the number of transfusions in
patients scheduled for major surgery (except heart surgery).2
The major experiences with these novel agents are from
their use in nephrology and oncology. Anemia in patients of
both the specialities results in poor quality of life, increased
cardiovascular risks and reduced long term survival.3 ESA
therapy has transformed the management of anemia in
these patient populations and has considerably reduced the
number of red cell transfusions.
Adverse Effects of Erythropoiesis
Stimulating Agents
All ESAs are effective in correcting anemia and increasing
hemoglobin levels but their different molecular properties
affects their clinical use. The routes of administration, the
financial considerations, the stage of disease, etc. all play
an important role in clinical decision making. ESA dose
 Chapter 58  Erythropoiesis Stimulating Agents versus Red Cell Transfusion  207
requirements are difficult to predict, but the target rate
of correction of anemia has been calculated as <1 g/dL to
2 g/dL per month. When hemoglobin level exceeds the target,
the ESA dose should be reduced without discontinuing the
treatment so as to avoid the excessive decrease in hemoglobin.
There is evidence that risk for mortality is high in patients
with chronic kidney disease when the hemoglobin levels
were persistently below 11 g/dL or levels that had significant
fluctuations between the target range and <11 g/dL.4 Thus
these agents have generated a ray of hope of an improved
outcome in an otherwise debilitating disease.
Despite the promising results, the overall effect of
therapy with ESA on patient outcome is still debated. This is
probably attributed the documentation of side effects such
as hypertension, seizures, possible cardiovascular events,
vascular access thrombosis, pure red cell aplasia and iron
deficiency.
Hypertension
Erythropoietin induced increase in hematocrit in nor­
motensive subjects is associated with a significant increase
in resting mean arterial pressure of 6 mm Hg as measured
by intra-arterial catheter. This has been attributed to
impaired endothelium dependent vasodilatation, systemic
vasoconstriction and renal mechanisms (hyporeninemic
hypoaldosteronism).5 In patients on hemodialysis, the
incidence of hypertension correlated with the dose of
erythropoietin. Intravenous erythropoietin doses of 40, 80
and 120 U/kg thrice weekly for 49 weeks were associated with
hypertension in 28%, 32% and 56% of subjects respectively.6
Similar results have been obtained with Darbepoetin.7 In
rare cases, hypertensive encephalopathy and seizures have
occurred. Blood pressure response differs markedly in RBC
transfusion where blood pressure decreases despite similar
increase in hematocrit. Most of the trials have under reported
the incidence of hypertension with ESAs due to standard of
care escalation of antihypertensive medications during the
trial. Also there has been a question mark over the quality
of trials as in most of the study groups, ultrafiltration during
dialysis was increased in line with increase in hematocrit.
Cardiovascular Complications
Randomized trials in patients undergoing dialysis have
found higher rates of cardiovascular and thromboembolic
complications with higher (13–15 g/dL) vs lower
(10–11.5 g/dL) hemoglobin targets.8-10 Two recent trials:
cardiovascular risk reduction by early anemia treatment
with epoetin beta (CREATE) and correction of hemoglobin
and outcomes in renal insufficiency (CHOIR), suggest that
targeting higher than recommended hemoglobin levels with
ESAs poses a safety risk.11,12 The Normal Hematocrit Study13
of patients with kidney and heart disease also identified a 1-
to 5-fold increased risk of myocardial infarctions and vascular
access thrombosis when ESAs were administered with target
hemoglobin levels of 14 g/dL. Around 2004, it became
apparent that as a result of treatment with ESA’s, patients with
end stage renal disease and cancer chemotherapy induced
anemia were having thromboembolic events. In a review of
trials (both independent and industry sponsored) evaluating
the role of ESAs for treatment of anemia, it was found that
there is 1.57-fold increased VTE risk and a 1.10-fold increased
mortality risk when ESAs were administered to patients with
anaemia and cancer.14
In another systematic meta-analysis addressing the
benefits and harms of ESAs for anemia, it was concluded that
the risk of serious adverse events was significantly higher
among recipients of ESAs than among controls.15 There is
inconclusive evidence to suggest that the use of ESAs in
treating anemia in patients with heart failure is beneficial.16
Limited evidence suggests that the presence of clinically
evident cardiac disease (congestive heart failure or ischemic
heart disease) in ESA-treated patients increased their risk
of nonfatal myocardial infarction. Consequently, extreme
caution should be used when using these drugs in patients
with established heart disease.
In November 2009, the results from the trial to reduce
cardiovascular events with aranesp therapy (TREAT) were
presented.17 Participants were randomly assigned into
receiving darbepoetin for a hemoglobin target of 13 g/dL, or
receiving placebo with rescue darbepoetin treatment below a
hemoglobin of 9 g/dL. There was a significant improvement
in quality of life but a doubling in the risk of stroke in the
normal hemoglobin arm provided support for an adverse
relationship between ESAs and stroke.17
Therefore, the use of ESAs should be carefully reconsidered
in patients with high risk of thromboembolic events such
as previous history of thrombosis, surgery, prolonged
immobilization and in patients with hypercoaguble states.
There is no data on preventive use of anticoagulants or
aspirin.
Pure Red Cell Aplasia
An extremely rare but serious adverse effect of the long-
term administration of ESAs is pure red cell aplasia reported
primarily with SC administration. Pure red-cell aplasia was
the most common adverse event associated with the use of
epoetin therapy in the database of the FDA’s Adverse Event
Reporting System. The production of immunoglobulin (Ig)
G antibodies against endogenous erythropoietin has been
demonstrated in patients with chronic kidney disease.
Therefore, the resulting anemia is much more severe than
prior to the onset of therapy. Bone marrow examination
shows almost complete absence of erythroid precursors,
but normal white cell and platelet precursors. Any patient
who develops a loss of response, severe anemia, and low
reticulocyte count should be evaluated.18 No pure red cell
aplasia has been reported in cancer patients.
208  Section 2  Cellular Therapies
Iron Deficiency
Iron deficiency occurs in most patients during therapy
with ESAs because of the increase in erythropoiesis and
subsequent increase in iron demand. Iron supplementation
should therefore be considered for all patients, and iron
status should be closely monitored.
Tumor Progression
Anemia in cancer patients can be due to malignancy
infiltrating the bone marrow or it can occur as a result of
systemic chemotherapy used for treatment. Expression
of erythropoietin and erythropoietin receptors has been
demonstrated in a variety of human cancers. Erythropoietin
after binding to erythropoietin receptors initiates signals that
stimulate growth, inhibit apoptosis and induce differentiation
of erythroid progenitors to increase red cell mass.
Erythropoieitn stimulation of cancer cells in vitro activates
signal transduction pathways, including phosphatidylinositol
3-kinase-Akt and JAK-STAT (Janus Kinase-Signal Transducer
and Activator of Transcription). The original rationale
for using ESA in cancer patients arose from evidences of
improved quality of life, reduction in transfusion dependency
and potential improved treatment efficacy. However, several
publications in the recent years have proposed ESAs may in
fact adversely affect patient survival. Recent trials such as
enhance (head and neck cancer), EPO-CAN 20 (nonsmall
cell lung cancer) GOG 191 (cervical cancer) and trials in
breast cancer have raised concerns over ESA therapy.19 These
trials reported shorter progression free survival in patients
treated with ESA. In fact a study on breast cancer (BEST)
was terminated prematurely due to increased incidence
of mortality in ESA arm.20 In another study conducted
on anemic patients with multiple types of cancer who
had not received chemotherapy/radiation, patients were
randomized to receive blood transfusions alone or ESA.
Survival was shortened in patients who received ESA. This
was not chemotherapy associated anemia but was anemia
associated with cancer itself.21 Although the cause of this
decreased survival is unknown, it has been speculated that
administration of ESA may cause tumor progression.22,23
Another hypothesis is that because of activation of signal
transduction pathways, efficacy of chemotherapy regimens
designed to target anti apoptotic pathways may be diminished
by ESAs.
Miscellaneous
Other side effects of ESAs are rare allergic reactions, skin
rash, urticaria, anaphylaxis, arthralgias, peripheral edema
and mild and transient injection site pain. Caution is also
recommended in patients with abnormal liver function tests.
RED CELL TRANSFUSION
In chronic anemias such as due to renal failure, cancers,
thalassemias, etc; there is increase in content of 2, 3 DPG with
a shift towards right in the hemoglobin dissociation curve
and there is increase in cardiac output and respiratory rate
as compensatory mechanisms. Therefore, physiologically it is
rarely necessary to transfuse such patients with hemoglobin
levels more than 8 g/dL. However, the impact of anemia in
cancer patients has been reported in terms of quality of life
and cancer treatment outcome. There is a higher incidence of
fatigue in anemic cancer patients. Also patients with different
tumor types have poor outcomes when they are anemic.
Around 15% of chronically anemic patients are treated
with RBC transfusion.24 Each unit of RBC with volume of
300 mL contains 200 mL RBCs and increases the hemoglobin
by 1 g/dL.
Complications of Transfusions
Blood transfusions come with risks such as procedural
problems, iron overload, possible viral and bacterial
infections, immune injury and immunomodulation.
Availability of High Quality Blood Products
It is a major problem when treating the anemic patient with
transfusions. After donation, blood units can be stored upto
42 days under stringent conditions. However, the quality of
product decreases during this period. Specific issues related
to storage of RBCs include changes in metabolism, shape, loss
of membrane proteins and alterations in oxygen delivery.25
Acute Hemolytic Reactions
Majority of hemolytic reactions are due to ABO
incompatibility. Commonly these are due to human errors
such as transfusion of blood to wrong patients or improper
identification of pre transfusion blood sample.
Nonimmune hemolysis during storage or administration
can occur due to physical disruption, temperature changes or
contact with nonisotonic fluids.
Transfusion Associated Circulatory Overload
Patients with cardiopulmonary disease, renal failure and
infants are at a high risk of volume overload especially during
rapid transfusion.
Iron Overload
One unit of RBCs contain 200 mg iron and this is released
into circulation following metabolism of hemoglobin from
 Chapter 58  Erythropoiesis Stimulating Agents versus Red Cell Transfusion  209
the transfused RBCs. Excess iron is typically present in liver,
heart, skin and endocrine organs. Organ toxicity begins when
reticuloendothelial sites of iron storage become saturated.
Therefore, continuous transfusion therapy in absence of iron
chelation can lead to fatal liver/heart dysfunction.
Infectious Disease Transmission
Current RBC transfusions are relatively safe in regards to
transfusion related infections. Viral transmission of HIV 1
and 2, HTLV, HBV and HCV have been limited by systematic
testing of blood products.26 Bacteria may be introduced into
the pack at the time of blood collection from sources such as
donor skin, donor bacteremia or equipment used for blood
processing. This risk is also minimal with RBCs as they are
stored in refrigerator where most of the contaminants are
nonviable.
Immune Injury
Acute immune injuries have emerged as the leading
complication of RBC transfusion. Most of these reactions
occur immediately after transfusion.
• Hemolytic transfusion reactions: They are caused by
pre-existing antibodies. Delayed hemolytic transfusion
reactions are seen 3–10 days after transfusion of apparently
cross matched compatible RBCs. In such cases, there has
been previous allo-immunization to minor RBC antigens,
therefore a history of prior transfusion or pregnancy is
mandatory.
• Febrile nonhemolytic transfusion reactions: It is defined as
1°C rise in temperature during or soon after transfusion.
It is attributed to reaction between recipient antibodies
and white cell antigen in the blood product. This too has
declined with use of leucodepletion techniques.
• Allergic blood transfusion reactions: It is seen in 1–3% of
RBC transfusion recipients. Anaphylaxis with hypotension,
bronchospasm, stridor, tachycardia, arrhythmias and GI
symptoms occur with one in 20,000–50,000 transfusions.
• Transfusion related acute lung injury (TRALI): It develops
within 2–8 hours of transfusion in patients without other
risk factors for acute lung injury. It is very rare but an
important cause of transfusion related mortality.
• Post transfusion purpura: It is a rare but serious
complication. There occurs destruction of platelets due to
antibodies against platelet specific antigens.
• Transfusion related graft vs host disease: A rare
complication, it occurs 8–10 days post transfusion. It
carries a mortality rate of 80–90% resulting from infections
and bleedind secondary to pancytopenia and liver
dysfunction. It is related to immune response mediated
by donor T lymphocytes against the recipient lymphoid
tissue.27
Impact of RBC Transfusion in Cancer
In immunocompromized patients, blood transfusion
may increase the risk for non-Hodgkin lymphoma. Also
there is increased relative risk for development of Chronic
lymphocytic leukemia/small lymphocytic lymphoma.28
Several retrospective studies looked at impact of RBC
transfusion on treatment outcome in patients with regional
cancer. Most of these studies had a curative intent (surgical
treatment) and revealed higher perioperative mortality in
patients receiving RBC transfusions.29
There is paucity of data on the rate of progression of
primary tumor when RBC transfusion is used as a mode of
correcting anemia.
Miscellaneous
Other complications include hypothermia, citrate toxicity,
hyperklemia etc.
CONCLUSION
Anemia in chronic diseases is associated with a significant
reduction in quality of life. Adequate management of
anemia is essential to the core treatment plan. Both
RBC transfusion and ESAs are efficacious means to
improve hemoglobin levels. However, recent data
indicates that ESAs, are associated with serious adverse
effects and have greater morbidity and mortality as
compared to RBC transfusion. There is insufficient data
to suggest improvement in health related quality of life in
chemotherapy induced anemia. The primary role of ESAs,
currently, is to decrease the number of transfusions and
not replace the same. Robust pharmacovigilance programs
are essential and are expected to shed additional light on
the effects of ESAs on survival and disease progression in
such patients.
references
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younger than ever. Eur J haematol. 2007;78(3):183-205.
2. http://www.fda.gov/drugs/DrugSafety/DrugSafetyPodcasts/
ucm077123.htm.
3. Locatelli F, Pisoni RL, Combe C, et al. Anaemia in haemodialysis
patients of five European countries: Association with morbidity
and mortality in the Dialysis Outcomes and Practice Patterns
Study (DOPPS). Nephrol Dial Transplant. 2004;19:121-32.
4. Eckardt KU, Kim J, Kronenberg F, et al. Haemoglobin variability
does not predict mortality in European hemodialysis patients. J
Am Soc Nephrol. 2010;21:1765-75.
5. Lundby C, Thomsen JJ, Boushel R, Koskolou M, Warberg J,
Calbet JA, et al. Erythropoietin treatment elevates haemoglobin
concentration by increasing red cell volume and depressing
plasma volume. J Physiol. 2007;578(Pt 1):309-14.
210  Section 2  Cellular Therapies
6. Pollok M, Bommer J, Gurland HJ, Koch KM, Schoeppe W,
Scigalla P, et al. Effects of recombinant human erythropoietin
treatment in end-stage renal failure patients. Results of a
multicenter phase II/III study. Contrib Nephrol. 1989;76:201-11.
7. Kanbay M, Akcay A, Delibasi T, Uz B, Kaya A, Koca C, et al.
Comparison of effects of darbepoetin alfa and epoetin alfa
on serum endothelin level and blood pressure. Adv Ther.
2007;24:346-52.
8. Furuland H, Linde T, Ahlmen J, Christensson A, Strombom U,
Danielson BG. A randomized controlled trial of haemoglobin
normalization with epoetin alfa in pre-dialysis and dialysis
patients. Nephrol Dial Transplant. 2003;18:353-6.
9. Besarab A, Bolton WK, Browne JK, et al. The effects of normal as
compared with low hematocrit values in patients with cardiac
disease who are receiving hemodialysis and epoetin. N Engl J
Med. 1998;339:584-90.
10. Parfrey PS, Foley RN, Wittreich BH, Sullivan DJ, Zagari MJ,
Frei D. Double-blind comparison of full and partial anaemia
correction in incident hemodialysis patients without
symptomatic heart disease. J Am Soc Nephrol. 2005;16:2180-9.
11. Drueke TB, Locatelli F, Clyne N, and CREATE Investigators.
Normalization of haemoglobin level in patients with chronic
kidney disease and anaemia. N Engl J Med. 2006;355:2071-84.
12. Singh AK, Szczech L, Tang KL, and CHOIR Investigators.
Correction of anaemia with epoetin alfa in chronic kidney
disease. N Engl J Med. 2006;355:2085-98.
13. Besarab A, Bolton WK, Browne JK, et al. The effects of normal as
compared with low hematocrit values in patients with cardiac
disease who are receiving hemodialysis and epoetin. N Engl J
Med. 1998;339(9):584-90.
14. Bennett CL, Silver SM, Djulbegovic B, Samaras AT, Blau CA,
Gleason KJ, et al. Venous thromboembolism and mortality
associated with recombinant erythropoietin and darbepoetin
administration for the treatment of cancer-associated
anaemia. JAMA. 2008;299:914-24.
15. Tonelli M, Hemmelgarn B, Reiman T, Manns B, Reaume MN,
Lloyd A, et al. Benefits and harms of erythropoiesis-stimulating
agents for anaemia related to cancer: a meta-analysis. CMAJ.
2009;180:E62-71.
16. Lindguist DE, Cruz JL, Brown JN. Use of erythropoiesis
stimulating agents in the treatment of anaemia in patients of
systolic heart failure. J Cardiovasc Pharmacol Ther. 2014; pii
1074248414541841 [Epub ahead of print].
17. Pfeffer MA, Burdmann EA, Chen CY, Cooper ME, de Zeeuw D,
Eckardt KU, et al. A trial of darbepoetin alfa in type 2 diabetes
and chronic kidney disease. N Engl J Med. 2009;361:2019e32.
18. Bennett CL, Luminari S, Nissenson AR, et al. Pure red cell
aplasia and Epoetin therapy. N Engl J Med. 2004;351:1403-8.
19. Dicato M, Plawny L. Erythropoietin in cancer patients: pros
and cons. Curr Opin Oncol. 2010;22:307-11.
20. Leyland-Jones BEST investigators and study group. Breast
cancer trial with erythropoietin terminated unexpectedly.
Lancet Oncol. 2003;4:459-60.
21. Glaspy J, Smith R, Aapro M, et al. Results from a phase 3,
randomized, double-blind, placebo-controlled study of
darbepoetin alfa for the treatment of anaemia in patients not
receiving chemotherapy or radiotherapy. Paper presented at
the American Association for Cancer Research Annual Meeting
Proceedings; April 14-18, 2007; Los Angeles, CA.
22. Bohilus J, Schmidlin K, Brillant C, Schwarzer G, Trelle S,
Seidenfeld J, et al. Recombinant human erythropoiesis
stimulating agents and mortality in patients with cancer: a
meta analysis of randomized trials. Lancet. 2009;373:1532-42.
23. Aapro M, Osterwalder B, Scherhag A, et al. Epoetin-beta
treatment in patients with cancer chemotherapy induced
anaemia: the impact of initial haemoglobin and target
haemoglobin levels on survival, tumor progression and
thromboembolic events. Br J Cancer. 2009;101:1961-71.
24. Crémieux PY, Barrett B, Anderson K, et al. Cost of outpatient
blood transfusion in cancer patients. J Clin Oncol. 2000;18:
2755-61.
25. Hess JR. Red cell storage. J Proteomics. 2010;73:368-73.
26. Bihl F, Castelli D, Marincola F, et al. Transfusion-transmitted
infections. J Transl Med. 2007;5:25.
27. Anderson KC, Weinstein HJ. Transfusion-associated graft-
versus-host disease. N Engl J Med. 1990;323:315-21.
28. Castillo JJ, Dalia S, Pascual SK. Association between red
blood cell transfusions and development of non-Hodgkin
lymphoma: A meta-analysis of observational studies. Blood.
2010;116:2897-907.
29. Vamvakas EC, Blajchman MA. Deleterious clinical effects of
transfusion associated immunomodulation: Fact or fiction?
Blood. 2001;97:1180-95.
Natural Killer Cells in Tumor
and Transplantation
Sunil K Arora, Rajendra Kumar
Existence of natural killer (NK) cells was first observed in 1975,
when it was shown that the body contains a small population
of lymphocytes that display cytotoxic activity against a wide
range of tumor cells even in the absence of any previous
sensitization with tumor antigen.1,2 Since their inception,
NK cells envisaged a fascinating transmutation from so
called ‘dumb, nonspecific killer cells’ to highly sophisticated
and well primed detectives for harmful changes in cellular
organization.
Natural killer cells are predominantly large granular
lymphocytes, and majority of them express CD16 and CD56
molecules as cell surface receptors. NK cells capable to kill
a variety of cells including virally infected cells and tumor
cells are known to promote graft rejection. NK cells constitute
5–10% of lymphocytes in human peripheral blood, and do not
express the T and B lymphocyte lineage markers. NK cells can
mediate cytolysis in the absence of major histocompatibility
complex (MHC Class I or II) antigen expression on their
target cells and as tumor cells are known to express low levels
of Class I MHC molecules, NK cells serve an important role in
anti-tumor immune response.
Origin and Distribution of
Natural Killer Cells
Past literature does not pinpoint about the precise lineage
of NK cells. However, several studies suggest that NK cells
originate from bone marrow precursors. CD34+ bone marrow
progenitors can be differentiated into NK cells, using in vitro
culture manipulation. Moreover, NK cells are shown to express
CD7 marker as an early event in NK cell lineage commitment.
Although, direct role of thymus in differentiation of NK cells
is not supported by the literature, thymic stromal cells shown
to support the NK cell differentiation from CD34+ progenitor
cells in vitro. In humans, NK cells usually are defined as CD3–
CD56+ lymphocytes and comprise of 5–20% of peripheral
blood lymphocytes. NK cells can further be divided into
two major subsets, called, CD56dim CD16+ and CD56bright
59
CD16–. The CD56dim population predominantly present in
the peripheral blood and has high cytotoxic potential, while
CD56bright reside mainly in the lymph nodes and produces
cytokines upon activation.3 Mouse is one of the common
models used to study many cancers; however, the mouse NK
cells do not express CD56. Recently, it has been identified
that NK cells in humans and mice can be defined by the
expression of NK receptor NKp46, which is almost exclusively
expressed by all NK cells in both species.4
Mechanism of Cytotoxicity
Natural killer (NK) cell mediates lysis of target cells within
minutes of exposure and this cytolysis can be considered to
occur in four stages: (1) Binding to target cell (Adhesion);
(2) Effector cell activation (Recognition); (3) Delivery of
lethal signal to target cell (Lethal hit); and (4) Detachment of
effector cell and recycling (Fig. 1). NKR-P1, being a prototype
identified in rats, now identified as receptor which mediates
adhesion to target and subsequent activation.5 As NKR-P1
is constitutively expressed by normal tissue, its ubiquitous
existance suggests the presence of inhibitory mechanisms to
protect normal tissues and cells from NK cell mediated lysis.
Based on prevention hypothesis, LY-49 in mouse and p58 and
NKB1 in humans have been identified as inhibitory receptors
to control over-ridden activated NK cells.6
Figure 1  Natural killer cell response to target cell
212  Section 2  Cellular Therapies
Role of Natural Killer Cells in
Transplant Immunology
Conventionally ‘transplantation immunology’ has focused
on adaptive component of immune system, i.e. T- and B-cell
reactions. Only recently natural killer (NK) cells have been
found to play an important role after transplantation of
solid organs and hematopoietic stem cells. Transplantation
process, results in tissue injury and inflammation, and as
a result, upon reperfusion of the graft, the beneficiary’s
immune system come across a plethora of soluble and cell
surface danger- and stress-signal molecules.
Natural killer (NK) cells have activating receptors which
recognize “cell stress” induced by various foreign activities.
These activating receptors are negatively regulated by the
interaction between inhibitory molecules and autologous
human leukocyte antigens (HLA). With the availability of an
array of activating receptors specific for stress-related cellular
events, NK cells are exclusively equipped to detect and react
to the damage initiated by injury. NK cell activation is a
phenomenon which includes coordination of all activating
and inhibitory signals received by the cell.7 The two major
components of this system are:
1. Inhibitory signals received by major histocompatibility
complex (MHC) class I molecules.
2. Activating signals received by molecules up regulated in
response to stress and specific to the NK cell lineage.
The activating NK cell receptor, i.e. NKG2D, recognizes
stress- or pathogen-derived ligands, whereas inhibitory killer
immunoglobulin-like receptors (KIR receptors) recognize
self, but not allogeneic human leukocyte antigens (HLA)-A,
-B, or -C. NK cell subsets express either KIR receptors or
CD94/NKG2A. KIRs are highly polymorphic whereas NKG2A
are less polymorphic.8
Although NK cell mediated organ damage might be
limited, the role of these cells as initiators or perpetuators of
adaptive immune responses, well known in other context,
has not received much attention in the transplant setting. The
stress sensor system of NKG2D-mediated activation has the
potential to override MHC class I inhibition in vitro, suggesting
the importance of understanding the consequences of ROS
injury on NK cells. As we all know, MHC system is inherited,
similarly the repertoire of KIR genes is also inherited, and
out of all KIR genes, certain genetic patterns are disease-
associated. The clinical challenge of infection in the setting
of immune suppression after transplantation may be as
important in the setting of cell therapies as it is in organ
transplantation, further highlighting the need to understand
NK cell responses for optimal transplant therapies.
Interestingly, many studies have shown that the natural
killer (NK) cells are also involved in immunity both against
residual tumor cells and against pathogens in patients
undergoing stem cell transplantation. Most importantly, the
beneficial effects attributed to NK cells in the post-transplant
period appear to occur without the risk of graft-versus-host
disease, which makes these cells uniquely suited for adoptive
immunotherapy.9 Unlike B- and T lymphocytes, NK cells do
not express clonally rearranged receptors to detect antigens.
Instead, activation is regulated by integration of signaling
from germ-line encoded activating and inhibitory cell surface
receptors.10
Over the past 20 years, NK cells have attracted
substantial interest in the context of hematopoietic stem cell
transplantation. While many aspects of NK cell biology in this
setting remain poorly understood, donor selection criteria
in HLA-mismatched transplantation include the potential
of a donor to generate alloreactive NK cells post-transplant.
Retrospective studies showing a survival advantage for
patients transplanted from an HLA-identical donor carrying
activating KIR receptors have led to large prospective trials
that aim to preferentially recruit stem cell donors with a
beneficial activating KIR gene profile. While these are exciting
times for researchers interested in NK cells, much work
remains to be performed to unravel the many ways in which
NK cells influence the transplant outcome.
Natural Killer Cells and Cancer
Surveillance
Myriad of reports from mouse model evidence the role of
NK cells in the abolition of tumor cells and removal of NK
cells functional arm often led to a more aggressive tumor
growth and metastasis in a tumor induced mouse and higher
frequencies of spontaneous tumor in chemical carcinogen
challenged mice. In humans, correlative studies show that
low NK cell activity was correlated with cancer risk which
indicates a role for NK cells in tumor surveillance.11 However,
unlike mouse model, low NK cells infiltration in established
tumors subverts the antitumor immune response. Low
infiltration which was attributed to inefficient homing into
malignant tissues could be meliorated by cytokine mediated
immune activation or by occurrence of alloreactive NK cells
provided by allogeneic hematopoietic stem cell transplant.12
In the recent past, NKG2D receptor has been identified
as a seminal NK cell receptor, which in concert with other
receptors like NKp30, NKp44 and NKp46, determine NK cell
activation toward tumor cells.
Natural killer (NK) cells play an important role in
protection against transplantable tumors. In varying range of
experimental models, this has been done by depletion of NK
cells before tumor challenge to show more aggressive tumor
growth. Other investigators incorporated the strategy of
administering cytokines enhancing NK cell effector functions,
e.g. IL-2, IL-12, IFN or IFN inducing agents to exhibit
enhanced antitumor activity. As a common observation, it
was noted from these experiments that NK cells are more
efficient at eliminating tumor deficient or low MHC-I on their
surface. Despite their well proven antitumor activity and
immune-surveillance, NK cells may not be able to completely
eliminate a tumor alone, but may seek cooperation from other
 Chapter 59  Natural Killer Cells in Tumor and Transplantation  213
arms of immune system such as T or B cells. Furthermore,
there is increasing evidence that indicate that although
NK cells alone may not be capable of eliminating tumors,
they may kill some tumor cells, providing apoptotic tumor
bodies to antigen presenting cells such as Dendritic cells, for
subsequent activation of T-cell response. Additionally, IFN-γ
or other cytokines secreted by NK cells in response to tumors
may activate local macrophages and dendritic cells, which in
return fuel NK cells by cell-cell contact or soluble factor in a
positive feed-back manner.13
Conclusion
Although identified as nonspecific killer cells, the field of
NK cells have gone through vast metamorphosis and this
developing area of investigation indicates that NK cells may
be more critical in facilitating immune response in a manner
that previously was not well appreciated.
References
1. Herberman RB, Nunn ME, Holden HT, et al. Natural cytotoxic
reactivity of mouse lymphoid cells against syngeneic and
allogeneic tumors. II. Characterization of effector cells.
International Journal of Cancer. 1975;16:230-9.
2. Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic
reactivity of mouse lymphoid cells against syngeneic and
allogeneic tumors. I. Distribution of reactivity and specificity.
International Journal of Cancer. 1975;16:216-29.
3. Freud AG, Caligiuri MA. Human natural killer cell development.
Immunological reviews. 2006;214:56-72.
4. Walzer T, Jaeger S, Chaix J, et al. Natural killer cells: from CD3−
NKp46+ to post-genomics meta-analyses. Current opinion in
immunology. 2007;19:365-72.
5. Trinchieri G. Biology of natural killer cells. Advances in
immunology. 1989;47:187-376.
6. Giorda R, Weisberg E, Ip T, et al. Genomic structure and strain-
specific expression of the natural killer cell receptor NKR-P1.
The Journal of Immunology. 1992;149:1957-63.
7. O’Connor GM, Hart OM, Gardiner CM. Putting the natural
killer cell in its place. Immunology. 2006;117:1-10.
8. Spahn JH, Li W, Kreisel D. Innate immune cells in
transplantation. Current opinion in organ transplantation.
2014;19:14-9.
9. Terszowski G, Passweg JR, Stern M. Natural killer cell immunity
after transplantation. Swiss Med Wkly. 2012;142:w13700.
10. Passweg J, Stern M, Koehl U, et al. Use of natural killer cells
in hematopoetic stem cell transplantation. Bone marrow
transplantation. 2005;35:637-43.
11. Wu JD, Higgins LM, Steinle A, Cosman D, Haugk K, Plymate
SR. Prevalent expression of the immunostimulatory MHC
class I chain–related molecule is counteracted by shedding
in prostate cancer. The Journal of Clinical Investigation.
2004;114:560-8.
12. Esendagli G, Bruderek K, Goldmann T, Busche A, Branscheid
D, Vollmer E, et al. Malignant and non-malignant lung tissue
areas are differentially populated by natural killer cells and
regulatory T cells in non-small cell lung cancer. Lung Cancer.
2008;59:32-40.
13. Piccioli D, Sbrana S, Melandri E, et al. Contact-dependent
stimulation and inhibition of dendritic cells by natural killer
cells. The Journal of Experimental Medicine. 2002;195:335-41.
Potential and Hazards
of Gene Therapy
Ajit C Gorakshakar
Human gene therapy to cure hereditary disorders is a topic
of public interest, because genetic diseases account for a
significant amount of human sufferings. More than 3000
genetic diseases are known to cause from defects in single
gene. Many such diseases are well known like sickle cell
anemia, thalassemias, cystic fibrosis, etc. while other diseases
like severe combined immunodeficiency syndromes, the
various lipid and carbohydrate storage diseases are less
common.
Gene therapy is the process where the abnormal or disease
causing gene is corrected at DNA level. The term Gene Therapy
was first introduced at the International Congress of Genetics
(Yaron et al. 1997). This can be done by actually repairing
the defect of the endogenous gene (targeting a gene) or by
introducing new genetic material in to the genome.
The actual process depends on the following variables:
• The disease to be treated
• The gene to be inserted
• The target cells of insertion
• The vector used to transmit the gene
• The route of administration of the gene.
Selection of a disease to work on is very important.
Generally single gene disorders with recessive inheritance
were selected for this purpose. Therefore lot of work has
been done on gene therapy to cure sickle cell anemia or
β-thalassemia where β-globin gene is corrected to produce
normal β-chains and in appropriate quantity.
This can be done by two ways:
1. Germline gene therapy: In this sperm and ova are
genetically altered. This is done by injecting the corrected
gene directly into a fertilized egg at risk so all the cells in
the body have altered genetic makeup and the genetic
disease is permanently cured.
2. Somatic gene therapy: This refers to manipulation of body
cells other than germinal tissue. This is performed on fully
developed individuals. This technique does not place the
whole organism at risk if it fails. So, the experiments can be
repeated on the same organism.
60
Historical Aspect
If we define gene therapy as treatment of a disease by
genetic manipulation then treatment of an individual with
thyroid hormones in hypothyroidism or with steroids to treat
inflammation in colitis should be considered as gene therapy
because this involves certain genes being turned up or down
or switched on or off. However, today we consider gene
therapy as a procedure to correct defective genes to cure the
disease.
It can be done by following ways:
1. Replacing mutated gene which causes disease with the
healthy gene.
2. Inactivating or knocking out a mutated gene. The gene can
be turned off, so it no longer promotes the disease.
3. Introducing a new gene into body to fight a disease.
The first attempt to treat a human disease by introducing
new genetic material in to the cells was carried out in the
early 1970s by Dr Stanfield Rogers to treat argininemia
in three sisters. This was performed before the existence
of Ethics committees. In 1980 Dr Cline tried to treat two
European patients suffering with beta thalasseima. In each
case bone marrow removed from the patient was subjected
to treatment with DNA containing the normal hemoglobin
gene. Considering various hazards involved in the procedure,
ethics boards were constituted to critically evaluate the
protocol before starting the experiments.
Gene Therapy in Transfusion Medicine
Transfusion medicine is a branch of science involving
administration of cells or any other biologic material like
serum, clotting factors, etc. to treat patients. In gene therapy
new biological material is transfused in patient body to treat
specific disease. Therefore, it comes under the preview of
transfusion medicine. Hematopoietic cells are excellent
targets for gene therapy. They can be easily accessed and may
be included ex vivo with the vectors containing the genetic
repair material. Lastly the disease for which gene therapy
 Chapter 60  Potential and Hazards of Gene Therapy  215
strategies are developed, directly affect the transfusion
medicine department. For example if gene therapies for
thalassemia or sickle cell anemia are developing and patents
are cured, this will reduce the load in blood bank considerably.
Prerequisite for gene therapy
The complete human genome sequencing project has
significantly contributed to ‘Gene Therapy’ experiments.
Because this project gave us the information about all the
genes present in entire human genome. Before gene therapy
experiments are started, one should be aware of complete
knowledge of a disease gene, including it’s molecular
structure, function and regulation. This is required to provide
the best ‘replacement part’.
Gene delivery systems
The main challenge of Gene therapy is the difficulty of
transporting large, fragile and negatively charged DNA
molecules into the nucleus of the cell without degradation. So,
one has to create safe and efficient gene delivery vehicles. For
this purpose knowledge about complete understanding of the
interaction mechanism between the target cells and delivery
system is required. Similarly knowledge about intercellular
traffic and targeting mechanism is also very important.
Vector design
There are many aspects involved in engineering the vector
to be used for gene therapy. They are, specific disease to be
treated, the target cells and the therapeutic strategy. The
normal/corrected gene which will be inserted can provide a
functional form of a missing or detecting gene or it can act as
a suicide gene (in cancer) designed to kill the cell in which
it is expressed. In case of cystic fibrosis in which the disease
causing gene is nonfunctional, a functional gene is added
while in case of thalassemia the gene for normal product of
fully functional β-globin gene is inserted.
Viral delivery systems
They consist of viruses which are replication deficient but
can deliver DNA for expression. Adenoviruses, retroviruses,
Adeno Associated viruses and lentiviruses are used as gene
delivery vectors (Escors and Breckpot 2010). They have high
gene transfection efficiency but their residual viral elements
can cause insertional mutagenesis and immunological
problems. In 1980s gene transfer to mammalian cells
was feasible after the development of retroviral vectors
(Yaron et al. 1997). In 2006, melanoma was successfully
treated using retroviral vectors.
Adenoviruses
Nonenveloped, double stranded DNA viruses, widely used
as DNA transfer vector. They can infect dividing as well as
quiescent cells with equally high efficiency. They have broad
cell specificity. Majority of human population is exposed
to adenoviruses. So antibodies against them are already
present in our body. Therefore, these vectors are degraded
within 24 hours even before reaching the target cells. They
quickly activate immune cells which can affect the patients
health. Other disadvantages included short term expression,
transient expression of foreign genes and immunogenicity.
Retroviruses
Small RNA viruses, replicating through DNA intermediates.
The genome consists of very few genes required for virus
replication. While designing the vector, these genes are
detected and replaced by the therapeutic gene (up to
10 kb). Moloney marine leukemia virus (MMLV) is the most
common retrovirus used for designing vector. It’s limitations
are low vector titer, low transfection efficiency, particle
instability and inability to transduce nondividing post mitotic
cells. To overcome these problems, vectors derived from
lentiviruses have been constructed. Lentiviruses are one
type of retroviruses which can infect both dividing as well as
quiescent cells. This virus ensures long term expression and
efficient transfer without inflammatory responses.
Adeno Associated Viruses
Single stranded nonenveloped DNA viruses. They have low
risk of insertional mutagenesis ‘Suicide’ gene therapy is
feasible using this virus. It’s genome is small (4 kbp). They
are able to transduce a wide variety of cells regardless of
their stage in the cell cycle. They are able to permanently
implant their genes into a particular part on chromosome 19
of humans. However, the main drawback of this vector is its
size of the genome which significantly limits the amount of
genetic material that it can carry.
Lentiviruses
They can deliver a significant amount of viral RNA into the
DNA of host cells. They have the unique ability to infect non
dividing cells. So, today genetically engineered lentivirus
is widely used as a gene delivery vector. This has some
advantages over other vectors. They include high efficiency
infection of dividing and nondividing cells, long-term
stable expression of a transgene and low immunogenecity.
Lentiviruses have been successfully used for transfection of
diabetic mice with the gene encoding platelet derived growth
factor (PDGF) (Lee et al. 2005) and also correcting hemophilia
in mouse model, by expressing wild type platelet factor VIII
gene (Shi et al. 2007). They do not require degradation of the
nuclear membrane for integration.
NonViral Delivery Systems
They are alternate to viral based systems. They can be
classified according to method of preparation as physical
216  Section 2  Cellular Therapies
or chemical types. The most common physical methods are
microinjection, electroporation, ultrasound and gene gun.
In physical methods physical force is applied to increase the
permeability of the cell membrane. However, it may cause
tissue damage. Chemical methods utilize natural or synthetic
carriers to deliver genes into cells. Here polymers, liposomes,
cationic lipid systems are used (Prokop and Davidson
2007). Naked plasmid DNA coated with gold particles was
effectively introduced to cells using gene gun technique. Lot
of work is being done for developing effective physical and
chemical systems to deliver transgenes into the cell to provide
appropriate expression (Gao et al. 2007).
Nucleic acid vectors also come under this category. They
are generally developed as a therapeutic strategy to treat
cancer. Nucleic acid strategy includes antisense and RNA
interference (RNAi) mechanism. However, lot of hurdles
need to be overcome before it is used as a gene delivery system
(Zhang et al. 2012). Small interfering RNA (siRNA) is capable
of reducing expression of non target genes due to interaction
of the siRNA strand with a partially complementary site on
an “off target” mRNA. Secondly innate immune activation
via RNA is a significant undesirable side effect due to the
toxicities associated with excessive cytokine release and
inflammatory syndromes. Another challenge is stabilization
of RNA because naked RNA is rapidly degraded by nuclease
present in the serum. Similarly RNA lack specific tumor
targeting and are quickly excreted by kidney upon systemic
administration. To overcome these challenges special non-
viral vectors like Lipoplex or Lipopolyplex are designed
(Li and Huang 2006, Wolff and Rozema 2008).
Advances in Globin Gene Therapy for the
Treatment of Beta Thalassemia and
Sickle Cell Anemia
Sickle cell anemia and thalassemia are hereditary monogenic
disorders related to hemoglobin synthesis. Therefore, the
gene therapy to cure these disorders involves the transfer of
a regulated globin gene in autologous hematopoietic stem
cells. Various globin vectors have been tried but they do not
express beta-globin at the same level. So different vector copy
numbers are required to correct these disorders. Similarly
various factors are considered to design the globin vector
safety. These include providing therapeutic levels of globin
transgene expression with a minimal risk of insertional
oncogenesis and toxicity. The transgene expression should
be erythroid specific and stage specific differentiation.
To achieve these criteria lot of work was done (Sadelain
et al. 1995). Finally the therapeutic levels of hemoglobin
synthesis were achieved in the progeny of virally transduced
HSCs (Hematopoietic Stem Cells). In a pioneering work by
May et al. (2000) thalassemia was corrected in thalassemic
mice using specially designed lentiviral vector (TNS9). This
vector encodes beta-globin gene with deletion of cryptic
poly-adenylation site within intron 2 and is flanked by an
extended promoter sequence and the beta-globin 3" proximal
enhancer as well as large locus control region (LCR) elements
(3.2 kb) spanning HS-2, 3 and 4. The LCR elements contribute
to the enhancer activity. The combination of these control
elements was found to be the best among several (Rivella
et al. 2003a). Pawliuk et al. (2001) corrected HBS-antilles-D
Punjab phenotype and Berkley (BERK) sickle cell disease
mouse model using a lentiviral vector having enhanced
anti-sickling features. Insulators are genomic elements that
can shelter genes from their surrounding chromosomal
environment. They can act either by blocking the action of
a distal enhancer on a promoter or by acting as barriers that
protect the gene from silencing effect of heterochromatin.
The most characterized insulator sequence is the chicken
hypersensitive site 4 (cHS4) of the chicken beta-like globin
cluster. Malik et al. (2005) added cHS4 insulator to a globin
lentiviral vector encoding the HS2-3-4 LCR fragments. They
obtained high level beta-globin expression in cultured
erythroid cells as well as after xeno-transplantation in
immunodeficient mice. In addition, using a single copy
MEL cell clones a two fold increase in human hemoglobin
expression at the mRNA level was demonstrated by the same
group (Arumugam et al. 2007).
Regulatory issues
In principle, the applications of gene therapy are numerous,
extending from treating monogenic disorders like sickle
cell anemia, cystic fibrosis, to acquired disorders like AIDS,
Cancer, neurological diseases, etc. Gene therapy is being
considered here because no alternative option for treatment
of these disorders is available. Recombinant DNA technology
is used in this therapy. Use of this technology in vivo has
induced public fears and speculation regarding potential
risks or dangerous side effects caused to it.
This technology involves production of genetically
engineered living cells using new biological product like
vectors. Among these vectors, defective viruses harbor the risk
of generating replication competent helps viruses resulting in
further spread. So the regulatory aspects can be addressed
along three lines.
1. Experimental and preclinical research.
2. Manufacturing of safe gene therapy products like vectors.
3. Clinical trial and further development of the subject.
More specifically the following aspects need to be
considered critically while starting gene therapy experiments:
Identification of gene causing the disease, its regulation, the
molecular basis of the diseases, development of gene delivery
system with potential biohazards, efficacy and safety outcome
following therapeutic treatment of the patient.
Several countries have formed high powered committees
to address these issues. They study the gene therapy
experiment protocol very carefully and critically and if found
suitable give their nod to perform experiments (Cohen-
Haguenauer 1997).
 Chapter 60  Potential and Hazards of Gene Therapy  217
Future of Gene Therapy
Over the years some solutions have been developed to
overcome various difficulties faced by scientists in 1990 when
experiments on gene therapy were started. The vectors have
been designed to yield optimal expression of the normal gene.
Guidelines related to gene therapy have been standardized
and followed carefully by various Ethic Boards to give
sanction to conduct clinical trials. Up to 2012 more than 1800
gene therapy trials have been completed while 1843 trials are
being conducted in 31 countries (Ginn et al. 2013).
The first successful gene therapy trial for beta-thalssemia
was reported by Cavazzana-Calvo et al. (2010) using lenti-
globin vector. They could achieve ‘transfusion independence’
situation in one adult patient suffering with severe HbE/
β0 thalassemia. However, the problems of genome toxicity,
oncogenesis and safety harbor of gene still persist. In case of
diseases like thalssemia where different molecular lesions are
responsible for the same phenotype, optimization of vector
copy number, the level of transgenic hemoglobin required to
correct each patient’s phenotype are different and work needs
to be done on this aspect. Nonviral vectors are potentially
safe to genetically modify the cells but there is a difficulty in
maintaining long term stable expression (Jackson et al. 2006).
Induced pluripotent stem cells are prepared by introducing
four genes viz. Oct-4, SOX-2, KLF4 and Myc into fibroblasts.
These cells are capable of differentiation in a way that is very
similar to embryonic stem cells which can grow into fully
differentiated tissues. Ye et al. (2009) published an article
suggesting that iPSCs might provide a possible new approach
to treat beta-thalassemia and sickle cell anemia. The
technology is very simple. First autologous iPSC lines can be
generated from fibroblasts of monogenic disorders. They are
corrected in vitro with wild version of the gene, differentiated
and transplanted back into diseased host. The corrected cells
should restore the disease free phenotype. One of the main
obstacles involves the need to eliminate the diseased cell
population from the host prior to transplantation. As Myc
gene is oncogenic, protocols have been developed to produce
iPSCs without the use of Myc gene (Nakagawa et al. 2008).
Xu et al. (2009), in a mouse model of hemophilia A,
differentiated iPSCs in vitro into endothelial cells which were
injected into the liver, where production of soluble factor VIII
was started in vivo. In 2007, Hanna et al. showed the use of a
targeted gene correction approach to cure Sickle cell anemia.
In this iPSCs were generated from mouse cells and the
mutated gene was substituted with wild type gene producing
normal hemoglobin by homologous recombination (HR).
The corrected cells were differentiated into hematopoietic
progenitors and subsequently transplanted into irradiated
animals thereby alleviating the diseased phenotype.
Correction of patient’s iPSCs by HR technique has some
potential advantages over conventional gene therapy. The
main advantage is that the risk of insertional mutagenesis by
therapeutic vectors is avoided. However, the main limitation
if it is to be applied to correct hemoglobinopathies is that
red cells derived iPSCs have limited ability to express adult
hemoglobin and ‘to home’ to the bone marrow.
Challenges in Gene Targeting in
Human Stem Cells
Random integration of transgene is generally used for
modifying genomes. However, such transgenes rarely
recapitulate the expression pattern of their endogenous
counterparts. Further it can cause insertional mutagenesis
which can lead to malignancy. The following techniques are
being used to overcome these challenges.
• The use of helper dependent Adenoviruses which have
highest DNA delivery efficiency and very low cytotoxocity.
• Introduction of double strand breaks to target diseased
loci. They are highly recombinogenic and can stimulate
homologous recombination.
For this purpose zinc finger nuclease (ZFN) and mega
nucleases (MGN) are being engineered and developed for
gene targeting. Today these custom designed molecular
‘scissors’ are being used for basic research and gene
correction experiments in iPSCs. They can also be used to
engineer cells which are resistant to chemotherapy. It can be
achieved by targeted addition of drug resistance transgene
(Sancho-Martinez et al. 2011). However, while doing this,
more thorough approaches are required to ensure the safety
of genome editing technologies.
Suggested reading
1. Arumugam PI, Scholes J, Perelman N, et al. Improved human
beta globin gene expression from self-inactivating lentiviral
vectors carrying the chicken hypersensitive site-4 (cHS4)
insulator element. Mol Ther. 2007;10:1863-71.
2. Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusion
independence and HMGA2 activation after gene therapy of
human beta thalassaemia. Nature. 2010;467:318-22.
3. Cohen-Haguenauer O. Gene therapy: Regulatory issues and
international approaches to regulation. Current Opinion in
Biotechnol. 1997;8:361-9.
4. Escors D, Brecpot K. Lentiviral vectors in gene therapy: their
current status and future potential. Archivum Immunologiae
et Therapia Experementalis 2010;58:107-19.
5. Gao X, Kim KS, Liu D. Non viral gene delivery: What we know
and what is next? Am Assoc Pharmaceuti Sc J. 2007;9:92-104.
6. Ginn SL, Alexander IE, Edelstein ML, et al. Gene therapy clinical
trial worldwide to 2012- an update. J Gene Med. 2013;15:65-77.
7. Hanna J, Wernig M, Markoulaki S. Treatment of Sickle Cell
Anemia mouse model with iPS cells generated from autologous
skin. Science. 2007;318:1920-3.
8. Jackson DA, Juranek S, Lipps HJ. Designing non viral vectors
for efficient gene transfer and long term gene expression. Mol
Ther. 2006;14:613-26.
9. Lee JA, Conejero JA, Mason JM, et al. Lentiviral transfection
with the PGDF-B gene improves diabetic wound healing. Plast
Reconstr Surg. 2005;116:532-8.
218  Section 2  Cellular Therapies
10. Li SD, Huang L. Targeted delivery of antisense oligo­
deoxynucleotide and small interference RNA into lung cancer
cells. Mol Pharm. 2006;3:579-88.
11. Malik P, Arumugam PI, Yee JK, et al. Successful correction of the
human Cooley’s anemia (beta) thalassaemia major phenotype
using a lentiviral vector flanked by the chicken hypersensitive
site 4 chromatin insulator. An NY Acad Sc. 2005;1054:238-49.
12. May C, Rivella S, Callegari J, et al. Therapeutic hemoglobin
synthesis in beta thalassemic mice expressing lentivirus
encode human beta globin. Nature. 2000;406:82-6.
13. Nakagawa M, Koyanagi M, Tanabe K, et al. Generation of
induced pluripotent stem cells without Myc from mouse and
human fibroblast. Nature Biotechnol. 2008;26:101-6.
14. Pawliuk R, Westerman KA, Fabry ME, et al. Correction of Sickle
Cell Disease in transgenic mouse models by gene therapy.
Science. 2001;294:2368-71.
15. Prokop A, Davidson JM. In Lanza R, Langer R, Vacanti J (Eds),
Gene delivery into cells and tissues. Principles of Tissue
Engineering. Elsevier Academic Press, ABD 2007. pp. 493-515.
16. Rivella S, Lisowski L, Sadelain M. Globin gene transfer: a
paradigm for transgene regulation and vector safety. Gene ther
and Regulation. 2003;2:149-75.
17. Sadelain M, Wang CHJ, Antoniou M, et al. Generation of a high
titer retroviral vector capable of expressing high levels of the
human beta globin gene. PNAS. 1995;92:6728-32.
18. Sanco-Martinez I, Li M, Belmonte I. Disease correction the
iPSC way: Advances in iPSC based therapy. Clin Pharmacol
and Therapeutics. 2011;89:746-9.
19. Shi Q, Wilcox DA, Fahs SA, et al. Lentivirus mediated platelet
derived factor eight gene therapy in murine hemophilia A.
J Thromb Hemost. 2007;5:352-61.
20. Wolff JA, Rozema DB. Breaking the bonds: Non viral vectors
become chemically dynamic. Mol Ther. 2008;16:8-15.
21. Xu D, Alipio Z, Fink LM. Phenotypic correction of murine
Hemophilia A using an iPSC based therapy. PNAS. 2009;106:
808-13.
22. Yaron Y, Kramer RL, Johnson MP, et al. Gene therapy: Is the
future here yet? Obstet Gynecol North Am. 1997;24:179-99.
23. Ye L, Chang JC, Lin C, et al. Induced pluripotent stem cells offer
new approach to therapy in thalassemia and sickle cell anemia
and option in prenatal diagnosis in genetic diseases. Proc Natl
Acad Sc USA. 2009;106:9826-30.
24. Zhang Y, Satterlee, Huang L. In vivo gene delivery by non viral
vectors : overcoming hurdles? Mol Ther. 2012;20:1298-304.
 Section

3
Blood Safety
Leucoreduction

Kanchan Bhardwaj
61
Leucoreduced blood product/leucoreduction (LR) are the Clinical Indications for
usage of blood products from which the leucocytes have been
removed. Leucoreduced red blood cells and platelets have Leucoreduction
been used to decrease the adverse effects of their transfusion
occurring because of allogenic donor leucocytes. It involves Established Indications
the process which removes leucocytes from the blood • Reduce frequency and severity of recurrent febrile non­
components and reduces the incidence of adverse events hemolytic transfusion reactions (FNHTRs) in chronically
associated with the donor leucocytes (Table 1). transfused patients.
• Reduce risk of CMV transmission to susceptible recipients
Approximate leucocytes in various (neonates, transplant recipients, oncology patients, etc.).
• Reduce rate of HLA alloimmunization and platelet
Red blood cell preparations refractoriness among hematology-oncology patients.
Blood components Number of leucocytes
Fresh WB ≈ 109 Potential Benefits of Leucoreduction
PRBCs ≈ 108 – 109 • Reduce risk of transfusion related immuo-modulatory
effect on cancer recurrence and postoperative bacterial
Buffy coat depleted PRBC ≈ 108
infection
Washed RBCs ≈ 107 • Reduce risk of transfusion associated graft-vs-host disease
Leucofiltered ≈ < 5 × 106 (TA-GvHD)
• Reduce organ dysfunction and mortality especially in
Critical antigenic load is the level of leucoreduction patients undergoing cardiac surgery1
to prevent FNHTR is 5 × 108 is one log reduction. It can be • Mortality rates are lower when Leucocyte-depleted blood
achieved by buffy coat removal. is used
Critical immunogenic load is the level of leucoreduction to • Reduction in storage lesions
prevent alloimmunization to HLA antigens or preventing the • Improved chance of finding an organ transplant match
transmission of lymphotrophic viruses the level of leucocyte • Possible reduction of variant Creutzfeldt–Jacob Disease
should be less than 5 × 106, i.e. 3 log reduction. (vCJD) transmission.

Table 1: Worldwide standards for leukodepletion in blood components

Organization Packed red blood cells Platelets
American Association of <5× 106 WBC/unit (red cell loss <15%) < 5 × 106 WBC/unit (apheresis platelets) with
Blood Bank >3 × 1011 platelets
< 8.3 × 105 WBC/unit (whole blood derived platelets)
European Council <1 × 106 WBC/unit (red cell loss <15%)
Director General of Health <5 × 106 WBC/unit (red cell loss <15%)
Services, India Out of 1% products, 75% should meet
the standards
222  Section 3  Blood Safety
• Possible reduction in transfusion related acute lung injury
(TRALI). The transfused antibodies, cytokines, etc. in the
blood unit react with the reciepient’s leucocytes leading to
a sequence of events that increase the permeability of the
pulmonary vasculature resulting in pulmonary edema.
Use of leucocyte reduction for all cellular components is
controversial. Based on the program of Hemovigilance in
France, several countries adopted universal leucodepletion
(ULD) over the potential spread of transmissible spongiform
encephalopathy by donor leucocytes in UK, and for general
reorganization of blood transfusion service in Canada.2,3 In
USA, ULD is not mandated by FDA. In India only less than 1%
of blood is leucoreduced.4
Preparation of Leucoreduced Blood
Specialized technologies are used to prepare the leucoreduced
products.
Methods of Leucodepletion
• Leucofiltration
• Apheresis
• Centrifugation and buffy coat removal
• Washed red cell concentrates
• Frozen deglycerolized red cells.
Leucofiltration
Most widely used method where 3rd and 4th generation
filters (leukocyte filter, 3–4 log leucoreduction (99.99%)
< 5 × 106 are used. The filters are attached to the blood bag and
the transfer bag and filtration is done by hanging the bags.
In-line leucodepletion filter is part of the blood collection
system. Prestorage leucodepletion can be done during blood
collection and processing. This is preferred in universal
leucodepletion. Leucoreduction filters are intended for a
particular component being transfused. The bag should hang
no longer than 8 hours.
Factors Affecting Leucofiltration
• Temperature of filtration is inversely proportional to the
efficiency of the filter
• Speed of blood flow through the filter is inversely
proportional to the filter efficiency
• Number of leucocytes presented to the filter effects its
performance
• Protein content of the suspending medium affects
leucoreduction
• Use of rinse step after filtration leads to spill over of
leucocytes
• Storage of blood at lower temperature improves perfor­
mance of leucofiltration
• Presence of HbS in the RBCs decreases the efficiency of
leucofiltration.
Mechanism of Filtration
Barrier filtration and cell adhesion (of negatively charged
leucocytes to the filter material by Vander Waals and
electrostatic force).
• Barrier filtration: Effective pore size of the order of 4 µm,
the space is sufficient for platelets and deformable RBC’s.
At higher temperature the leucocytes are deformable
so the filter performance decrease for RBC’s at room
temperature than for refrigerated RBC’s.
• Cell adhesion: For cell adhesion to occur there should
be sufficient dwell time of the blood within the medium.
High flow rates, nature of fluid in which the cells are
suspended, including the plasma protein content and the
platelet count affects the adhesion of leucocytes. It has
been demonstrated that the presence of viable platelets
improves the performance of leucofiltration.5
• Leucofiltration of platelet concentrates poses challenge
because of platelet adhesiveness. To counter the platelet
adhesiveness because of von Willebrand’s factor-
mediated platelet adhesion, the surface chemistry of the
filter media is modified to decrease the binding of platelets
to the filter.6 The leucocyte content of an entire unit of
leucoreduced RBC is equal to the leucoreduced content of
only one drop (100 µL) of non leucoreduced blood.
Biophysical Reasons for Reduced Performance
of Leucofiltration
The reduced efficiency of leucofiltration is due to following
reasons:
• Temperature—reduced efficiency of leucofiltration is
applied to higher temperature.
• Shear force—excess shear force results in cell detachment.
• Post filtration rinsing of filters result in leucocyte
detachment.
• Excessive volume of blood or blood containing excessive
number of leucocytes can overwhelm the filter capacity
from the filter medium.
• Presence of sickle cell trait in the donor RBCs can result in
reduced efficiency of filter. This is because of reduced cell
deformability of AS (sickle trait) RBCs under reduced oxygen
tension and pH within the filter. These RBCs either directly
clog the filter or reduce the effective area of filtration.
Timing of Leucofiltration
• Prestorage filtration
• Poststorage filtration
• Bedside filtration.
Prestorage Filtration
The filtration is done during or within 72 hours of preparation
before any leucocyte breakdown. So, no leukocyte breakdown

products are formed. So, it prevents FNHTR, CMV transmis­
sion, HLA alloimmunization and better quality control main­
tenance can be done (Table 2).
Poststorage Filtration
The filtration of components prior to issue from the laboratory
or at the bedside of the patient prior to transfusion. During
storage leucocytes breakdown and cytokines accumulate
which are not removed (Table 2).
Low Leucocyte Apheresis Devices
• Based on differences in cell mass of cells to be separated.
• Interface detection system–achieve standard leucodeple­
tion.
• The Amicus apheresis system used for preparing platelet
concentrates. The device incorporate three features:
active interface; autoelutrition and fluid flow dynamics.
An optical interface detector is positioned within the
separation chamber to monitor changes in the platelet
interface.
• Trima accel automated system separates platelets from
donor whole blood using a centrifugal technology with
saturated fluidized particle—bed dynamics to achieve a
natural separation of WBC’s from platelets.
Centrifugation and Buffy Coat Removal
It is done by automated component extractions. This gives a
log 1 reduction. It is enough to prevent FNHTR. It does not
prevent HLA alloimmunization/viral transmission. As per the
definition of LR, this cannot be labeled as LR. In developing
countries like India, this is used for reducing FNHTRs.
Washing
The red blood cells are washed by using normal saline,
resulting in open system hence limiting the shelf life. Not
used for preparation of leucoreduced packed red cells.
Table 2: Prestorage leucodepletion vs poststorage leucodepletion
Prestorage leucodepletion*
Timing of Done soon after collection
leucodepletion
Safety Safer (WBC removed before fragmentation)
Advantages Fewer transfusion adverse events
QA High quality process control
Techniques used Lab-side filtration/inline filtration/apheresis
Filters Removes problems associated with clogging of filters
Nursing time Saves nursing time
Disadvantages Needs prior planning and inventory management
*Prestorage leucoreduction is preferred
Chapter 61  Leucoreduction  223
Freezing and Deglycerolization
It is a costly and time consuming procedure so not used for
such preparations. It is reserved for storing rare blood groups
cells.
Quality Control of Leucodepletion (Table 3)
No common standards exist for all nations. The American
Association of Blood Bank Standards (AABBS) require that
95% of the units sampled meet the requirement for residual
leucocyte content.7 In Europe, the threshold requirement
for leucocytes is <1 × 106/unit. Techniques available for
counting methods specifically designed for the extremely low
concentration of white blood cells found in leucoreduced
blood components are microscopy, flow cytometry, and
the polymerase chain reaction.8 The relative proportion of
residual leukocyte subpopulations in leucoreduced blood
is not addressed by any standards. Different leucocyte
subpopulations are relevant for different biological effects
attributed to donor leucocytes. The clinical outcomes
evidence of a particular leucoreduction technique is superior
to any other for the removal of specific population is not
there.9,10 All the methods of leucoreduction that meet the
threshold standard for total number of residual leucocytes are
considered equivalent. Process problems have been reported
with both leucofiltration and apheresis. Kao11 et al. repor-
ted that 7% of apheresis platelets and 5% pooled platelets
failed and for RBCs 0.3–2.7% of units failed. They also reported
substantial losses of platelets and RBCs during the process of
leucofiltration. In apheresis the technique can fail. Factors
like sudden change in rate of flow entering the machine or
pauses in blood flow disturb the centrifugal separation of
blood resulting in spillover of leucocytes into the platelets.
Febrile Nonhemolytic Transfusion Reactions
It is unexplained rise in temperature of 1°C. There may
be only rigors only without rise in temperature, so early
onset of fever is not required for the diagnosis. Febrile
Poststorage leucodepletion
Done many days after collection, just before release
from lab or at bedside before transfusion
WBC fragments present
More transfusion reactions due to leukocyte fragments
Quality difficult to maintain
Lab-side or bedside filtration
Cell filters get clogged
Nursing time is more
Customized so no inventory management problems
224  Section 3  Blood Safety
nonhemolytic transfusion reactions (FNHTRs) develop after
the discontinuation of the transfusion in 10–20% cases and
mostly occur towards the end of the transfusion. The reactions
are dose related and require time to develop. These are quite
debilitating and occur in very ill, chronically transfused
patients. The incidence of FNHTR depends on the component
transfused. Non-leucoreduced RBCs are associated with 0.5%
to 0.6% risk of FNHTR, whereas platelet concentrates carry
20–30% risk. While with leucoreduced RBCs the FNHTRs are
< 0.5% and with platelets 1–4%.12
Mechanism of Febrile Nonhemolytic
Transfusion Reactions
The FNHTR is caused by three mechanisms:
1. Donor cell cytokines: RBC and platelet concentrate
transfusion result in this mechanism. In this recipient’s
anti WBC antibodies interact with transfused donor WBC.
Hence are prevented more effectively by prestorage LR
than bedside LR.
2. Recipient cytokines: Seen much more in platelet concen­
trate than in RBC transfusion. It results from the formation
of immune complexes between recipient’s antibodies
and antigens on donor cell/proteins present in the
transfused unit trigger release of cytokines by recipient’s
macrophages. They are prevented more effectively by
prestorage LR for donor WBCs but not for donor platelets.
3. Passive cytokine transfer/infusion: Seen in platelet concen­
trate transfusion. The cytokines are generated during room
temperature platelet storage. It is prevented by prestorage
LR only.
Clinical Evaluation of Febrile Nonhemolytic
Transfusion Reactions
Leucoreduction is highly effective in preventing FNHTRs to
RBCs in chronically transfused patients (thallassemia).13,14
RBCs do not accumulate clinically important levels of cytokines
because of refrigerated storage of RBCs as compared to platelets.
These reactions are rare with properly leucoreduced RBCs. In
case FNHTR occurs, these patients should be evaluated for
the presence of hemolytic reactions due to RBC blood group
incompatibility and for the presence of bacterial contamination
Table 3: Showing quality control of leucoreduction
Product Standards
Leucoreduced packed red Residual leucocytes < 5 × 106
blood cells (LR PRBCs) Red cell loss < 15% of original
Leucoreduced apheresis Residual leucocytes < 5 × 106
platelets/single donor Total platelet count ≥ 3 × 1011
platelets (LR SDP) pH 6.2
Leucoreduced random Residual leucocytes < 5 × 105
donor platelets (LR RDP) Total platelet count ≥ 4.5–5.5 × 1010
pH 6.2
of the transfused product. While FNHTRs in case of platelet
concentrates are much more common and are not completely
eliminated by LR. Patients experiencing FNHTRs to platelet
concentrates should be evaluated for platelet increments,
for evidence of alloimmunization, for drug-induced platelet
refractoriness, for bacterial contamination of platelets, and for
passive transfer of alloantibodies to ABO antigens from donor
to recipient.
CMV Transmission and Leucocytes
It is a leukotropic virus. High risk recipients for transfusion
acquired CMV are-low birth infants and immunosuppressed
patients. 80% population–seropositive for CMV, routine test­
ing is not effective. It stays as latent infection in mononuclear
cells. Leukodepleted–seronegative blood should be given to
susceptible patients.
HLA Alloimmunization
Donor leucocytes express HLA antigens. They sensitize
recipient to HLA alloantigen. Prevention is valuable for
bone marrow/stem cell transplant patients; patients on
chemotherapy and multi transfused patients.
Adverse effects of filtration and
leucodepletion
• Cost factor
• Substantial loss of therapeutic blood components
intended for transfusion
• Hemolysis due to prestorage filtration
• Hypotensive reaction to bedside leucoreduction (due to
bradykinins and activation of the coagulation pathways
and contact system during passage of blood through
leucoreduction filters)
• Hypotensive reaction to prestorage leucoreduced compo­
nents—in patients on ACE inhibitors.
Universal leucodepletion vs Selective
leucodepletion
Universal leucodepletion is practiced in Europe and Canada.
It is not approved by FDA in USA.
Pros and Cons of Universal Leucoreduction
• Pros
- Patients with selected indications to receive leuko­
depleted units will more likely receive leucoreduced
units.
- Inventory management is streamlined.
• Cons
- Large clinical studies fail to demonstrate clear benefit
of leucoreduction on proposed transfusion–associated
immunosuppression.

- It adds the extra financial burden to the health care
cost.
- Affect the adequacy of blood supply and interfere with
the supply of blood donors of African ancestry (sickle
cell trait).
- It removes physician’s choice from product selection.
Leucodepletion—Indian Perspective
In India selective leucodepletion is done. It is done by
adopting Buffy coat method of component preparation which
gives 1 log reduction and can minimize FNHTR. In special
cases harvesting component through apheresis technology
and pre storage/post storage leucodepletion in special cases
like patients with repeated FNHTR, patients on regular trans­
fusion therapy, immunosuppressed patients–CMV negative
blood and neonates requiring exchange transfusion.
References
1. van de Watering LMG, et al. Circulation. 1998;97:562-8.
2. Vamvaks EC, Blajchman MA. Universal WBC reduction: the
case for and against. Transfusion. 2001;41:691-712.
3. Seftel MD. Universal prestorage leucoreduction in Canada
decreases platelet alloimmunisation and refractoriness. Blood.
2004;103(1):333-9.
4. Fisk JM, Snyder EL. Universal pre-storage leucoreduction—a
defensible use of hospital resource: the Yale-New Haven
Hospital experience. Dev Bio (Basel). 2005;120:39-44.
5. Steneker I. Leukocyte Depletion from Fresh Red Cell
Concentrates by Fiber Filtration: Filtration Mechanism.
Amsterdam, VU University Press, 1992.
Chapter 61  Leucoreduction  225
6. Nishimura T, Kuroda T, Mizoguchi Y, et al. Advanced methods
for leukocyte removal by blood filtration. In: Brozovic B (Ed).
The Role of Leukocyte Depletion in Blood Transfusion Practice:
Proceedings of the International Workshop. Oxford, Blackwell
Scientific, 1989. pp. 35-40.
7. Silva MA (Ed). Standards for Blood Banks and Transfusion
Services, 23rd edn. Bethesda, Md., American Association of
Blood Banks, 2005.
8. Van der Meer PF, Gratama JW, van Delden CJ, et al. Comparison
of five platforms for enumeration of residual leucocytes in
leucoreduced blood components. Br J Haematol. 2001;115:
953-62.
9. Triulzi DJ, Meyer EM, Donnenberg AD. WBC subset analysis
of WBC-reduced platelet components. Transfusion. 2000;40:
771-80.
10. Roback JD, Bray RA, Hillyer CD. Longitudinal monitoring of
WBC subsets in packed RBC units after filtration: implications
for transfusion transmission of infections. Transfusion. 2000;
40:500-6.
11. Kao KJ, Mickel M, Braine HG, et al. White cell reduction in
platelet concentrates and packed red cells by filtration: a
multicenter clinical trial. The Trap study group. Transfusion.
1995;35:13-9.
12. Sherrill J Slichter. Leukocyte Reduction and Ultraviolet B
Irradiation of Platelets to Prevent Alloimmunization and
Refractoriness to Platelet Transfusions. The Trial to Reduce
Alloimmunization to Platelets Study Group. N Engl J Med.
1997;337:1861-70.
13. Sirchia G, Rebulla P, Parravicini A, Carnelli V, Gianotti GA,
Bertolini F. Leukocyte depletion of red cell units at the bedside
by transfusion through a new filter. Transfusion. 1987;27:402-5.
14. Sirchia G, Wenz B, Rebulla P, et al. Removal of white cells
from red cells of transfusion through a new filter. Transfusion.
1990;30:30-3.
Improving Blood Donor Screening
by Nucleic Acid Technology
Rekha Hans
Annually, millions of people worldwide receive blood compo­
nents or blood derived products for different indications, so
safety of these products remains the major responsibility and
concern of any blood transfusing service. A single donation can
be transfused to three or more different patients which means
screening blood for transfusion transmitted infections (TTIs)
is a very crucial step before taking the blood units into the
inventory. Although screening of blood for these infections is
being practised worldwide, TTIs are still being transmitted and
reported though their incidence has decreased significantly.
Need for Nucleic acid technology
The conventionally used screening methods target the
human body’s response to a virus (virus induced antibodies),
so the interval between the donor’s exposure to a virus and
detectable amount of antibodies/antigens (window period)
is missed by these methods of screening, thus creating a
need for a more sensitive and specific method to detect the
presence of virus directly. The German Red Cross Blood donor
service, Frankfurt took the lead to develop its first “in-house”
method of nucleic acid testing of transfusion transmissible
viruses. Now-a-days fully automated FDA certified methods
are available for nucleic acid technology (NAT) testing that
has been adopted in many countries for blood screening.
Nucleic acid technology
The method of NAT is based on amplification of targeted
regions of viral ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA).
The two commonly used NAT-screening technologies are
polymerase chain reaction (PCR), and transcription-mediated
amplification (TMA). PCR is a method that amplifies single
or small amounts of DNA segments, producing thousands or
millions of copies of specific DNA sequences from the target
sequences.1  TMA is a transcription-amplification process
that uses two enzymes—reverse transcriptase and RNA
polymerase to produce millions of copies of the targeted RNA
62
sequences. TMA consists of three main steps: target capture,
amplification, and detection.2  It allows for simultaneous
testing of multiple viruses in a single test tube, making the
assay convenient and faster.
Blood donor screening
Blood donor screening by NAT can be divided into four
groups3:
1. Transfusion relevant pathogens for which NAT testing is
done in most countries (HBV, HCV and HIV)—the HIV
virus has doubling time of approximately 17 hours. The
diagnostic window period of 15 days by conventional
method has been reduced to 8–9 days by screening with
mini pool of up to 96 samples per pool and can further be
reduced to 5–6 days for Individual Donation (ID-NAT).
Similarly, the diagnostic window period for HCV is 80
days for enzyme immunoassays and has reduced to 6–7
days with mini pool NAT ( pool size of 96) and further to
4–5 days with ID-NAT.
Compared to HIV-1 or HCV, the HBV virus has
doubling time of approximately 2.56 days.4 NAT has
helped in reducing the window period of HBV from
38.3 days with serological methods to 24.6 days (ID-NAT).3
The major risk of HBV transmission stems from occult
hepatitis B infections which harbours low viral load in
absence of seroreactivity to HBsAg. With NAT, the risk
of these infections has decreased and this has benefited
multi-transfused patients like thalassamics and those with
cancer. NAT also complements serological screening by
detecting false negative serology results.
2. Transfusion relevant pathogens for which testing is done in
some countries like United States for WNV. Other viruses
are HAV, B19V, Chickungunya virus and HIV-2. There are
cost effective issues which needs to be addressed before
recommending NAT for these viruses.
3. Probably transfusion relevant pathogens but not tested
as part of blood donor screening (HAV, SARS CoV and
Influenza virus).
 Chapter 62  Improving Blood Donor Screening by Nucleic Acid Technology  227
4. Bacterial screening by NAT—bacterial contamination
of blood products represents an on going challenge
in transfusion medicine and the product of concern is
platelets that are stored at room temperature. Many
countries have implemented culture methods such as
BacT ⁄ ALERT to detect bacterial contamination of platelets
but it has a long incubation time. The total screening time
for NAT systems (extraction and amplification) takes
approximately 4 hours.3 Therefore, this method offer
opportunities for a late sample collection before issuing
the platelets.
Nucleic acid technology is used for blood screening in
many countries like  Australia, Belgium, Brazil, Bulgaria,
Caribbean, Czech Republic, Denmark, Egypt, Estonia,
France, Germany, Greece, Hong Kong, Hungary, Indonesia,
India, Israel, Ireland, Italy, Korea, Latvia, Lithuania, Malaysia,
Mexico, Netherlands, New Zealand, Poland, Portugal,
Romania, Singapore, Slovenia, Slovakia, South Africa, South
Korea, Spain, Sweden, Switzerland, Thailand, Turkey, United
Kingdom, United States.
Germany was the first country to start NAT testing in
pooled sample size of 96 (Mini pool NAT) followed by other
countries. NAT is also available for testing each donation
individually (ID-NAT). This format of NAT seems more
sensitive as shown by data from many studies where ID-
NAT has been compared to MP NAT with pools of 16 or 8 or
4 samples.5,6
Experiences of different countries
United States
Nucleic acid technology was started in late 1990’s in US to
supplement serology testing, first for HIV and HCV, and
later for HBV and WNV. Today, 100% of the US blood supply
is screened with MP-NAT for HIV-1, HCV, WNV, and HBV
and it has helped to prevent the transmission of 5 HIV-1
infections and 56 HCV infections annually and has reduced
the residual risk of transfusion-transmitted HIV-1 and HCV
to approximately 1 in 2 million blood units.7
Germany
Nucleic acid technology for HCV was first introduced in
Germany in 1997 and it was performed on pooled samples
of 96 blood donations (Mini pool NAT). According to an
initial study8 a total of 31,524,571 blood donations (covering
approximately 80% of all the blood collected in Germany
during 1997 to 2005) were screened and 23 HCV, 7 HIV-1, and
43 HBV NAT-only–positive donations were detected.
United Kingdom
In United Kingdom, NAT has reduced the risk of HCV by 95%
and that of HIV by 10%.9
Italy
Nucleic acid technology implementation has improved
blood safety by reducing the risk of entering 2.5 HCV and
1.8 HIV infectious units per million donations into the blood
supply. The yield of NAT was higher for HBV than for HCV
or HIV. Over a study period of 6 years from 2001 to 2006, a
total of 10,776,288 units were tested for the presence of HCV
RNA, 7,932,430 for HIV RNA, and 3,405,497 for HBV DNA,
respectively. 2.5 per million tested were HCV RNA–positive/
anti-HCV–negative; 1.8 per million units tested were HIV
RNA–positive/ anti-HIV–negative; and 57.8 per million
donations tested were HBV DNA–positive/hepatitis B surface
antigen–negative.10
Hongkong
HBV NAT-yield rates for both WP and occult HBV infection
(OBI) increased significantly from 1:34,471 to 1:17,362 and
from 1:5120 to1:2450, despite a 1.2- and 1.6-fold decrease in
hepatitis B surface antigen (HBsAg) incidence and prevalence
rates respectively. Their current WP transmission risk with
NAT screening is 1:55,000 compared to 1:22,000 with HBsAg
testing.11
Japan
From February 2000 to April 2001, specimens from 6,805,010
units of serologically negative donation was screened in mini
pools of 50 samples by NAT using multiplex HBV/HCV/HIV-l
reagent. Among them, 112 HBV DNA-positives, 25 HCV RNA
positives and 4 HIV-l RNA positives were screened out and
they could prevent transfusion of this NAT positive units.12
China
In a pilot study of 18 months from China, ID NAT was
compared with enzyme immunoassays. It was observed
that HBV yield rate in their population is 1:1,056 for blood
donations.13 
Egypt
In a study from Egypt, 5 window period HCV donations were
identified among 15655 first time donors (yield 1:3100).14
Switzerland
The introduction of ID-NAT in Switzerland has reduced the
risk of HBV window period transmission in repeat donors
from 1 in 95,000 to 1 in 296,000.15
South Africa
In a study16 looking at the impact of ID NAT testing after
one year implementation. It was found that the HIV,
HBV, and HCV ID-NAT window phase yielded rates were
228  Section 3  Blood Safety
1:45,765, 1:11,810, and 1:732,200, respectively. The residual
transmission risk of ID-NAT HIV, HBV, and HCV window
phase donations was estimated at 1:479,000, 1:61,500, and
1:21,000,000 respectively.
Thailand
A study by Nantachit et al.17 evaluated 5,083 consecutive
blood donors in Chiang Mai, Northern Thailand for HIV-1,
HBV and HCV with NAT and compared these results to the
routine serologic screening for HBsAg, anti-HCV and anti-
HIV. They found that there were no NAT yield cases for HIV-
1 or HCV. There were 17 samples with discrepant HBV DNA
NAT and hepatitis B surface antigen (HBsAg) tests. Out of
these 17 samples, seven of the samples were HBV DNA NAT
positive but was HBsAg negative.
India
The First study was done by Makroo et al.18 where a total
of 12224 samples along with their serological results were
obtained from eight different blood banks in India and were
tested individually manually by procleix ultrio assay for
HIV-1, HCV and HBV. They observed eight NAT yield
cases. Study from western part of India showed combined
NAT yield (NAT reactive/seronegative) for HIV, HCV, and
HBV as 0.034% (1 in 2972 donations)19 which is high when
compared to studies from developed countries. In another
study conducted in north India, 18,354 donors were tested
by both ID-NAT and fourth generation ELISA, 7 were found
to be NAT-positive but ELISA-negative (NAT yield) for HBV
and HCV. The prevalence of NAT yield cases among routine
donors was 1 in 2622 donations tested (0.038%).20 In another
study from a tertiary care center from north India, ID NAT
results were compared to serological method for 73,898
samples, 1.49% were reactive by NAT, HIV-1 (0.09%), HCV
(0.25%), 1.05% were reactive for HBV only, and around 0.08%
were HBV-HCV co-infections with a combined yield of 1 in
610 donations (total 121 NAT yields).21 This high yield of NAT
is due to high prevalence of TTIs in India, further highlighting
the need for NAT in high prevalence countries to seek zero
transmission risk of TTIs.
Experience from many countries worldwide has proven
the efficacy of NAT in preventing TTIs and providing extra
layer of safety against these infections. However, NAT is
technically very demanding and involves issues of high costs,
dedicated infrastructure facility, equipments, consumables
and technical expertise. These issues are of particular concern
for resource limited developing countries where around 45%
of donations are not even screened using basic serological
methods.22 So before harping on universal NAT screening
for developing and 3rd world countries, the basic serological
testing of all donations in addition to adequate blood donor
screening, donor notification and counseling should be
ensured.
References
1. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT
et al. Primer-directed enzymatic amplification of DNA with a
thermostable DNA polymerase. Science. 1988; 239:488-91.
2. Giachetti C, Linnen JM, Kolk DP, Dockter J, Gillotte-Taylor K,
Park M, et al. Highly sensitive multiplex assay for detection of
human immunodeficiency virus type 1 and hepatitis C virus
RNA. J Clin Microbiol. 2002;40:2408-19.
3. Schmidt M, Seifried E. Improving blood donor screening by
nucleic acid technology (NAT) ISBT Science Series. 2010;5:
219-29.
4. Biswas R, Tabor E, Hsia CC, Wright DJ, Laycock ME, Fiebig EW,
et al. Comparative sensitivity of HBV NATs and HBsAg assays
for detection of acute HBV infection. Transfusion. 2003;43:788-
98.
5. Vermeulen M, Coleman C, Mitchel J, Reddy R, van Drimmelen
H,  Ficket T, et al. Sensitivity of individual-donation and mini
pool nucleic acid amplification test options in detecting
window period and occult hepatitis B virus infections.
Transfusion. 2013;53:2459-66.
6. Bouchardeau F, Girault A, Razer A, Servant Delmas A, Mercier
M, Laperche S. Sensitivity of Hepatitis B virus DNA transcription
mediated amplification testing in hepatitis surface antigen
positive blood donation. Transfusion. 2006;46:2047-52.
7. Stramer SL, Glynn SA, Kleinman SH, et al.  Detection of HIV-
1 and HCV Infections among Antibody-Negative Blood
Donors by Nucleic Acid–Amplification Testing. N Engl J Med.
2004;351:760-8.
8. Hourfar MK, Jork C, Schottstedt V, Weber-Schehl M, Brixner V
Busch MP, et al. Experience of German Red Cross blood donor
services with nucleic acid testing: results of screening more
than 30 million blood donations for human immunodeficiency
virus-1, hepatitis C virus, and hepatitis B virus. Transfusion.
2008;48:1558-66.
9. Soldan K, Davison K, Dow B. Estimates of the frequency of HBV,
HCV, and HIV infectious donations entering the blood supply
in the United Kingdom, 1996 to 2003. Euro Surveill. 2005;10:
17-9.
10. Velati C, Romanò L, Fomiatti L, et al. Impact of nucleic acid
testing for hepatitis B virus, hepatitis C virus, and human
immunodeficiency virus on the safety of blood supply in Italy:
a 6-year survey. Transfusion. 2008;48:2205-13.
11. Tsoi W, Lelie N, Lin C. Enhanced detection of hepatitis B
virus in Hong Kong blood donors after introduction of a
more sensitive transcription-mediated amplification assay.
Transfusion. 2013;53:2477-88.
12. Ohnuma H, Tanaka T, Yoshikawa A, Murokawa H, Minegishi
K, Yamanaka R, et al. The First Large-Scale Nucleic Acid
Amplification Testing (NAT) of Donated Blood Using Multiplex
Reagent for Simultaneous Detection of HBV, HCV and HIV-1
and Significance of NAT for HBV. Microbiol Immunol. 2001;45:
667-72.
13. Dong J, Wu Y, Zhu H, Li G, Lv M, Wu D, et al. A pilot study on
screening blood donors with individual-donation nucleic acid
testing in China. Blood Transfus. 2013;23:1-8.
14. ElEkiaby M, Laperche S, Moftah M, et al. The impact of
different HCV blood screening technologies on the reduction
of transfusion transmitted HCV infection risk in Egypt. Vox
Sang. 2009;96(suppl 2C-S08-03):23-4.
 Chapter 62  Improving Blood Donor Screening by Nucleic Acid Technology  229
15. Stolz M, Tinguely C, Graziani M, Fontana S, Gowland P,
Buser A, et al. Efficacy of individual nucleic acid amplification
testing in reducing the risk of transfusion-transmitted hepatitis
B virus infection in Switzerland, a low-endemic region.
Transfusion. 2010;50:2695-706.
16. Vermeulen M, Lelie N, Sykes W, Crookes R, Swanevelder J,
Gaggia L, et al. Impact of individual-donation nucleic acid
testing on risk of human immunodeficiency virus, hepatitis B
virus, and hepatitis C virus transmission by blood transfusion
in South Africa. Transfusion. 2009;49:1115-25.
17. Nantachit N, Thaikruea L, Thongsawat S, leetrakool N,
Fongsatikul L, Sompan P, et al. Evaluation of a multiplex human
immunodeficiency virus-1, hepatitis C virus, and hepatitis B
virus nucleic acid testing assay to detect viremic blood donors
in northern Thailand. Transfusion. 2007;47:1803-8.
18. Makroo RN, Choudhury N, Jagannathan L, Malhotra MP,
Chaudhary RK, Marwaha N. Multicenter evaluation of
individual donor nucleic acid testing (NAT) for simultaneous
detection of human immunodeficiency virus-1&hepatitis B&C
Viruses in Indian blood donors. Indian J Med Res. 2008;127:
140-7.
19. Jain R,  Aggarwal P, Gupta GN. Need for nucleic acid testing
in countries with high prevalence of transfusion transmitted
infections. ISRN Hematol. 2012;718671 Epub 2012 Sep 12.
20. Chatterjee K,  Coshic P,  Borgohain M,  Premchand,  Thapliyal
RM,  Chakroborty S,  et al. Individual donor nucleic acid
testing  for blood  safety against HIV-1 and hepatitis B and C
viruses in a tertiary care hospital. Natl Med J India.  2012;25:
207-9.
21. Agarwal N,  Chatterjee K,  Coshic P,  Borgohain M. Nucleic
acid testing for blood banks: An experience from a tertiary care
center in New Delhi, India. Transfus Apher Sci. 2013;49:482-4.
22. World Health Organization and International federation of Red
Cross and Red crescent societies. Safe blood starts with me.
World Health Organization. Geneva. 2000. p. 12.
ID-NAT Versus MP-NAT
Rajesh Kumar
Blood transfusion provides an essential and life saving support
in modern health care, when used appropriately, it saves
lives. However, it can cause acute or delayed complications
and also carries the risk of transfusion transmitted infections
(TTIs) such as human immunodeficiency virus (HIV-1),
hepatitis B virus (HBV), hepatitis C virus (HCV) and other
diseases. Despite the current practice of screening blood with
enzyme linked immuno sorbent assays (ELISA) of different
sensitivities and even after implementing the more sensitive,
newest generation of serological test, a considerable residual
risk of transfusion transmission of these virus remains.
Although the more sensitive serological tests have shortened
the pre-seroconversion window period, they still are not able
to identify a number of newly infected blood donors.
In spite of all the precautionary measures used to avoid
inoculating infective agents into transfusion recipients; it
is possible to transmit disease when blood from a recently
infected donor fails to be identified by routine screening
tests. This is because of the so called window period, i.e.
the period in which a donor is infected but before the
condition is detectable by routine methods. This technologic
limitation puts recipients at a tangible, albeit infrequent
risk for transmissible disease. Since viremia precedes sero
conversion by several days in case of HIV and several weeks
for hepatitis B and C virus, tests that detect viral nucleic acid
are considered a significant technologic and an addition
step in our quest to achieve the goal of zero risk for blood
transfusion recipients. Nucleic acid amplification test (NAT)
has the potential to detect viremia earlier than current
screening methods which are based on seroconversion.
NAT is currently being used in selected centers for donor
screening, though it is not mandatory by Drugs and Cosmetics
Rules. It is a highly sensitive method of testing blood. It is a
direct test which detects the viral ribonucleic acid (RNA) and
deoxyribonucleic acid (DNA) by the amplification method.
It reduces the window period by detecting low level of viral
genomic materials that are present soon after infection but
before the body starts producing antibodies in response to a
virus. This allows for earlier detection of infection and further
63
decrease the possibility of transmission via transfusion and
also detects mutants and occult cases.
Although NAT screening cannot completely eliminate the
risk of TTIs, but it has reduced the risk HIV, HCV and HBV
where it has been implemented. Implementation of NAT has
introduced not only a new methodology, new logistic but
when combine with sensitive serology it provides the most
sensitive and specific screening platform for blood screening.
The window period for detection by ID-NAT by ultrio plus is
4.7 days for HIV-1, 2.2 days for HCV and 14.9 days for HBV
and with pools of multiple (MP-6) donors is 11 for HIV-1 and
2, 12 days for HCV and 25 days for HBV. The corresponding
window periods for serological test are 15–20 days. 2–26 weeks
and 50–150 day respectively. The objective of NAT testing is
to provide blood in minimum risk to recipients through the
detection of as few viral copies (Cps) as possible of all known
genotypes, and sub groups. The NAT tests of high sensitivity
rely on amplification of intended target regions of viral nucleic
acids (NA) for detection. The aspects which influence the final
outcome of analytical sensitivity can be categorized under
three headings: (a) Sample preparation, (b) Amplification,
(c) Detection. In sample preparation the decisive steps are the
volume of infectious sample used for testing. The efficiency
of virus disruption to release NA, efficiency of NA extraction,
efficiency of NA elution if required, and removal of substances
that interfere in the following steps. The success in the
amplification category is ensured by the volume of extracted
NA used for target region amplification, and the ability to
tolerate interfering substances, the detection efficiency is
determined by the volume of amplicon used for detection,
the detection technology, the type and number of probes, and
the ability to demarcate signal from noise. The final output
of all these factors defines the analytical sensitivity. Nearly all
screening assays are qualitative assays scoring for reactives.
The analytical sensitivity also known as the limit of
detection (LoD) is determined through dilution analysis of
known standards, followed by probit analysis to predict the
minimum Cps/International Units (IU) that can be detected
at a particular frequency. The concentrations detected at

frequencies of 95% and 50% are typically used for defining
the LoD. Of great practical relevance is the screening
sensitivity of the NAT assay which is determined by the mode
of practice. Since serology tests are executed as individual
donation testes, this is a situation unique to NAT testing.
Currently NAT assays in blood screening are executed in the
format of (a) Individual donation-(ID), or (b) as multi/mini
pool (MP) donations. In ID-NAT testing, similar to serology
testing as aliquot of each individual sample is tested directly
and provides the screening sensitivity which is equivalent to
the analytical sensitivity. In MP-NAT testing predetermined
number of donations from as few as 4 to as large as 96 are
equally combined prior to testing. Based on the prevalence
of infection in the donor population, assuming that the other
members in the pool do not have virus, the virus concentration
of the original sample is diluted by the factor of the pool size.
The decreased concentration of the virions in the final testing
aliquot impacts the probability of detection. In other words
LoD is inversely related to pool size, with larger the pool size
greater the loss in sensitivity. Grabarczyk et al.1 observe that
the 95% LoD for HBV being 4 IU/mL for ID-NAT becomes
24 IU/mL for MP6; and for an ID-NAT assay with a LoD for
5 IU/mL becomes 120 IU/mL for MP24. Lin et al.2 report
that 50% LoD of 0.46 IU/mL for HBV ID-NAT translates to
1.56 IU/mL for MP6.
Several comparative studies evaluating ID-NAT versus
pool testing, using the same reagent, thereby excluding the
variability of reagent and platforms have been executed. Using
the Procleix Ultrio Plus assay Vermeulen et al.3 observed a
decrease in detection of HIV NAT yields as follows: 10% in
MP4, 20% in MP8, 30% in MP16. Grabarczyk et al.1 compared
HBV NAT yields for MP6 with Cobas TaqScreen MPX versus
Cobas Ampliscreen in MP24 and observed 12.6 folds increase
for HBV WP and 6.6 fold increases for occult hepatitis B
infection (OBI) NAT yields. In order to understand the
performance of the commercial reagents in the commonly
practiced format, the Procleix Ultrio Plus assay in the ID-NAT
format has been compared with the TaqScreen assay in the
MP6 format. For HIV-1 NAT yields the decrease in detection
from ID NAT with Procleix Ultrio Plus assay was 17% in pools
of 6 by Cobas Taqscreen assay. For HBV the overall decrease
was 30%, with more pronounced decrease during 2nd WP
characterized by low viral load (49% decrease) and OBI (33%
decrease) HBV infection status.4
The ability to successfully implement ID NAT in India and
its impact on interdicting release of infectious units has been
demonstrated in a multicenter study with centralized testing
and on-site screening studies.5,6 Although, the argument of
Chapter 63  ID-NAT Versus MP-NAT  231
affordability of ID-NAT has been the premise for promotion
of pool testing. The residual risk of the resulting TTIs places
a serious burden on the health-care, finances, society and
human life. HIV-2 from India was reported from Mumbai
in 1991; infections have been reported from several states of
India. HIV-2 was detected in high-risk groups and professional
blood donors. The seroprevalence of HIV-1 (Mono infection)
in a survey conducted in five urban and five rural populations
of Tamil Nadu was 0.1% while dual infection with HIV-1
and HIV-2 was 0.44%. The one of the drawback of Ulrio plus
ID-NAT is unable to detect the HIV-2 but detected by Cobas
TaqScreen assay in the MP6.
However, in India, high prevalence of TTI’s and compo­
nent transfusion is not widely accepted, the need for safe
blood is of utmost importance to the repeat transfusion
requiring group of patients with thalassemia, sickle cell
anemia, malignancy and hemophilia. For blood screening
the most sensitive assay in the most sensitive format should
be practiced to minimize the risk of TTIs.
References
1. Grabarczyk P, Letowska M, Kopacz A, Leszewski G, Sulkowska
E, Brojer E. Effectiveness of various minipool nucleic acid
test systems for hepatitis B virus detection in blood donors.
Transfusion. 2009;49:1494-5.
2. Lin KT, Chang CL, Tsai MH, Lin KS, Saldanha J, Hung CM.
Detection and identification of occult HBV in blood donors
in Taiwan using a commercial, multiplex, multi-dye nucleic
acid amplification technology screening test. Vox Sanguinis.
2014;106:103-10.
3. Vermeulen M, Coleman C, Mitchel J, Reddy R, Van Drimmelen
H, Fickett T, et al. Comparison of human immunodeficiency
virus assays in window phase and elite controller samples:
Viral load distribution and implication for transmission risk.
Tranfusion. 2013;53:2384-98.
4. Vermeulen M, Coleman C, Mitchel J, Reddy R, Van Drimmelen
H, Fickett T, et al. Sensitivity of individual-donation and
minipool nucleic acid amplification test options in detecting
window period and occult hepatitis B virus infections.
Transfusion. 2013;53:2459-66.
5. Makroo RN, Choudhury N, Jagannathan K, Parihar-Malhotra
M, Raina V, Chaudhary RK, et al. Multicenter evaluation of
individual donor nucleic acid testing (NAT) for simultaneous
detection of human immunodeficiency virus–1 and hepatitis B
& C viruses in Indian blood donors. Indian J Med Res. 2008;
127:140-7.
6. Agarwal N, Chatterjee K, Coshic P, et al. Nucleic acid testing
for blood banks: An experience from a tertiary care
centre in New Delhi, India. Tranfus Apher Sci. 2013;49:
482-4.
Occult Hbv Infection:
A Risk for Transfusion
Sheetal Malhotra
Introduction
Occult hepatitis B infection (OBI) is defined as the long-
lasting persistence of hepatitis B virus (HBV) genomes in
the liver (with detectable or undetectable HBV DNA in the
serum) of individuals testing negative for the HBV surface
antigen (HBsAg) in the presence (80%) or absence (20%) of
anti-HBc with or without anti-HBs antibodies.1 The amount
of HBV DNA in the serum is usually very low (<200 IU/mL) or
intermittently detected making detection of such infections
difficult but donation by such individuals is a potential
source of HBV transmission to recipients.2 It is emerging as a
challenging problem in blood transfusion safety.
Etiopathogenesis
Occult hepatitis B infection (OBI) may be observed in the
window period of acute HBV infection in blood donors and in
recipients of blood and blood products or in patients with HCV
chronic infection and in immunosuppressed individuals. It
shows the high prevalence in HCV co-infected patients as
HBV activity can be impaired by other infectious agents.3 OBI
is related to infection with variant viruses (S-escape mutants)
undetectable by the available HBsAg tests, but is more
often caused by the host mechanisms producing a strong
suppression of the HBV replication. It represents the fifth
phase in the natural history of chronic hepatitis B (HBsAg-
negative phase).4 Anti-HBc only/isolated anti-HBc positive
cases without anti-HBs is seen in resolving acute infection,
low grade chronic infection in the nonreplicative phase, in
case of infection with variant strains of HBV or due to false
positive anti-HBc reactivity.5
Epidemiology
Global Seroprevalence
The first recorded evidence of OBI and possible transmission
via blood transfusion is in late 1970s.6 Prevalence of OBI
varies in different studies due to differences in sensitivity of
64
the assay and endemicity of HBV infection (individuals from
countries highly endemic for HBV are more likely to develop
OBI).7 Various studies have been undertaken to estimate
seroprevalence of OBI in blood donors. A study from Egypt
showed a seroprevalence of OBI as 4.1%.3 A prospective study
undertaken on 31,190 first time voluntary blood donors from
Italy showed anti-HBc positivity in 8.3% samples of which
10.4% were ‘anti HBc only’. HBV DNA was found in 0.55% of
anti-HBc-positive/HBsAg negative subjects.2 Data from other
countries shows following figures –8/5973 donors or 0.13%
(with viral load <12u/mL) from Taiwan,8 3/600 or 0.5% donors
from Saudi Arabia,9 anti-HBc positivity in 42/100 donors of
which 90.5% were positive for HBV DNA from Sudan,10 16
OBI donors/131 anti-HBc positive donors (OBI 12.2%) from
Iran,11 anti-HBc positivity in 167/966 donors (17.28%) with
76 demonstrating anti-HBs positivity and 5 with detectable
HBV DNA from Pakistan,12 48 OBI donors/72 HBV DNA
reactive/HBsAg negative donors (66.7%) from Thailand,13 35
OBI donors/320 HBsAg-negative, anti-HBc and/or anti-HBs-
positive donors (10.9%) from Lao PDR,14 and 3.7% from US.15
Seroprevalence in India
Studies on Indian blood donors show an OBI seroprevalence
of 20–30% in anti-HBc positive donors. A study undertaken
by Bhattacharya et al. on 1027 HBsAg negative donors
revealed that 188 (18.3%) of them were anti-HBc positive, out
of which 21% were positive for HBV DNA.16 Another study
by Choudhary et al. involving 30,853 donors from Cohort 1
(24,694 in 2001) and Cohort 2 (6159 in 2000) were screened
for anti-HBc and anti-HBs. HBV DNA was detected in 40
out of 147 (27.21%) and 48 out of 230 (20.87%) donors with
anti-HBc only from the two cohorts.17 In a study by Hari
et al. from PGIMER Chandigarh, anti-HBc positivity was seen
in 8.4% (142/1700) of the blood donor samples. HBV DNA was
detected in 1 of 100 anti-HBc reactive donors tested. None of
the anti-HBc-only samples were positive for HBV DNA.18 In a
study by Doda et al. on 28,134 HBsAg Elisa negative samples,
HBV DNA was detected in 18 samples, out of which12 were
found to be OBI.19
 Chapter 64  Occult Hbv Infection: A Risk for Transfusion  233
Genotype characterization of OBI shows prevalence of
genotype C, B and a newly emerging genotype I in Thailand,
genotype D in Europe, genotype E in Ghana and genotype B
in China.20 In India, HBV genotype D is associated with high
occult HBV infections.21
Clinical Implications of OBI
The clinical implications of OBI involve different clinical
contexts as follows:
1. Risk of HBV transmission through, blood transfusion,
hemodialysis and organ transplantation.
2. Causing cryptogenic liver diseaseas immunosuppression
may induce OBI reactivation and development of acute
and sometimes fulminant hepatitis.
3. Contributing to the progression of liver disease and HCC.
4. Affecting response to antiviral therapy for patients with
chronic HCV infection, contributing to the progression of
the chronic liver disease toward cirrhosis.
Thus, OBI as a cause of chronic liver disease in patients
with HBsAg-negative status is becoming an important disease
entity. OBI has been reported as a potential risk factor for the
development of nonhepatic malignancies like pancreatic
cancer and hematological malignancies.4,22
Risk of TT HBV with OBI
HBsAg negative individuals with positive anti-HBc and/or
positive for antibodies against HBsAg may harbor HBV DNA.
Blood donation by such individuals is a potential source of
HBV transmission to the recipients. OBI may be involved
in the transmission of HBV by blood transfusion in two
conditions:
1. “OBI carrier”when the donor is infected with wild-type
HBV strains suppressed in the irreplication activity.
Chronic occult infection frequently shows fluctuating
levels of viremia (with fluctuating infectivity) with periods
when HBV DNA is not detectable in the serum.
2. The donor is infected with S-escape mutant HBV is able to
replicate but synthesize a mutated HBsAg not detected by
the routinely available diagnostic assays. This is the most
frequent condition associated with the residual cases of
hepatitis B related to blood transfusion.4
In a study from Australia, OBI residual risk was calculated as
approximately 1 in 982,000 per unit transfused. The infectivity
of components derived from donors with OBI depends on a
number of donor and recipient factors, particularly the anti-
HBs level in transfused components.23 The residual risk of
OBI estimated by Taira et al. in a study from Japan was 252/
million donations (1 in 3968 donations). They concluded that
severe hepatitis can occur by transfusion from OBI donors
despite the comparatively lower viral loads in components
and they identified two fatal post-transfusion fulminant HBV
cases associated with OBI donors in look back studies.24
Case reports by Lui et al. and Levicnik-Stezinan et al.
have shown that blood products from donors with an occult
HBV infection can result in TT HBV.25,26 A study in Taiwan
showed TT HBV transmission in two of 11 recipients of OBI
blood became subsequently infected.27 In another study from
the Japanese Red Cross, OBI blood was infectious in 19% of
recipients.28 In a prospective sudy involving 247 patients by
Tani et al. from Japan, the residual risk of blood transfusion
from donors with low anti-HBc titers or high anti-HBc titers
with high anti-HBs titers was evaluated and none of the
recipients showed reactivity for any of the HBV markers
(HBsAg, anti-HBs, anti-HBc and HBV DNA).29A study from
India on 174 multi transfused thalassemic patients, HBV DNA
was detected in 50% of patients with anti-HBc as the only
marker (OBI).30
Preventive Strategies
The above figures indicate that OBI is considerable risk in
transfusion recipients; this biological entity has implications
for donor and blood screening. Preventive strategies include
stringent donor screening including permanent deferral
of donors with a history of jaundice together with a broad
hepatitis B vaccination strategy.14,23 Symptomatic infection
with HBV may occur in approximately 30–50% of cases,
assuming this symptomatic infection rate also applies to OBI
cases, a strict deferral policy of donors with a history of hepatitis
or jaundice would exclude 30–50% of OBI donors.23 Anti-HBc
is a life-long serological marker of previous HBV infection, as
it is detectable both in acute and chronic infections and after
viral clearance; this marker, therefore, indicates a current or
past HBV infection. Screening for anti-HBc is recommended
in several countries in which the prevalence of hepatitis B is
low.2 Inclusion of anti-HBc antibody screening would exclude
the HBsAg negative/DNA-positive blood units; it would
exclude anti-HBc antibody positive blood with undetectable
HBV DNA leading to unnecessary deferral of blood donors
especially in countries with high HBV seroprevalence where
large number of blood donors are anti-HBc positive.14
The efficacy of the test is also limited by two main factors.
First is its lack of specificity. It is postulated that 70–90% of
samples containing high levels of anti-HBs antibodies are
essentially safe for transfusion unless transfused to immuno­
compromized recipients. Tani et al. from Japan concluded
in a study that blood components from donors nonreactive
for HBsAg and HBV DNA but reactive for anti-HBc titers of
<1:32 or anti-HBc titers of >1:32 with anti-HBs >200 mIU⁄ mL
are considered suitable for transfusion.29 Anti-HBc screening
also fails to identify two sources of anti-HBc negative
potentially infectious blood, in the pre-seroconversion
window period or late chronic infection referred to as “core
window period”.31As anti-HBc positivity is too high to allow
anti-HBc detection to be implemented on a routine basis in
our country, the use of HBsAg testing together with NAT can
234  Section 3  Blood Safety
be adopted as the policy of choice for screening donors in
order to further reduce and prevent HBV transmission. It is
postulated that samples collected in the post-acute phase of
infection often show very low levels of HBV DNA in the serum
(<100 IU/mL) and can, therefore, be missed when testing is
carried out in large pools or by less sensitive assays for HBV-
DNA.8 Requirement for NAT testing in such regions is an
assay that is very sensitive for the detection and identification
of HBV DNA and is especially useful in countries with a high
prevalence of HBV where occult HBV infections are quite
common and detection can be challenging. In a study by
Linet al from Taiwan, all 8 of NAT-yield cases were from
donors with an occult infection.8 Satoh et al. has introduced
a new method of concentrating HBV DNA and HBsAg
simultaneously to increase the sensitivity of detection tests.
By this method, 35 of the 77 anti-HBc-positive and HBsAg-
negative donors were HBV DNA-positive by ID NAT and
a further five donors became HBV DNA-positive by HBV
concentration. Twenty-seven of 40 occult HBV infections
became HBsAg-positive byHBsAg concentration.32 Still there
are few documented reports of HBV transmission by donors
missed ID NAT, so “zero risk” for HBV is yet to come.33
References
1. Raimondo G, Caccamo G, Filomia R, Pollicino T. Occult HBV
infection. Semin Immunopathol. 2013;35:39-52.
2. Romano L, Velati C, Cambie G, Fomiatti L, Galli C, Zanetti AR,
et al. Hepatitis B virus infection among first-time blood donors
in Italy: prevalence and correlates between serological patterns
and occult infection. Blood Transfus. 2013;11:281-8.
3. EL-Ghitany EM, Farghaly AG, Hashsish MH. Occult hepatitis
B virus infection among hepatitis C virus seropositive and
seronegative blood donors in Alexandria, Egypt. J Egypt Public
Health Assoc. 2013,88:18-31.
4. Squadrito G, Spinella R, Raimondo G. The clin; ical significance
of occult HBV infection. Annals of Gastroenterology. 2014. pp.
2715-19.
5. Gessoni G, Beggio S, Barin P, Favarato M, Galli C, Valverde S,
et al. Significance of anti-HBc only in blood donors: a
serological and virological study after hepatitis B vaccination.
Blood Transfus. 2014;12:s63-8.
6. Tabor E, Hoofnagle J, Small wood L, Drucker JA, Pineda-
Tamondong GC, Ni LY, et al. Studies of donors who transmitpost
transfusion hepatitis. Transfusion. 1979;19:725-31.
7. Emara MH. Occult hepatitis B: the Egyptian situation. Tropical
Gastroenterology. 2012;33:242-50.
8. Lin KT, Chang CL, Tsai MH, Lin KS, Saldanha J, Hung CM.
Detection and identification of occult HBV in blood donors
in Taiwan using a commercial, multiplex, multidye nucleic
acid amplification technology screening test. Vox Sang.
2014;106:103-10.
9. Bamaqa MS, Azahar EI, AL-Ghamdi AK, Alenzi FQ, Farahat
FM. Nucleic acid amplification technology for hepatitis B virus
and its role in blood donation screening in blood banks. Saudi
Med J. 2009;30:1416-21.
10. Mahmoud OA, Ghazal AA, Metwally Del S, Elnour AM,
Yousif GE. Detection of occult hepatitis B virus infection
among blood donors in Sudan. J Egypt Public Health Assoc.
2013;88(1):14-8.
11. Behzad-Behbahani A, Mafi-Nejad A, Tabei SZ, Lankarani
KB, Torab A, Moaddeb A. Anti-HBc & HBV-DNA detection in
blood donors negative for hepatitis B virus surface antigen in
reducing risk of transfusion associated HBV infection. Indian J
Med Res. 2006;123:37-42.
12. Bhatti FA, Ullah Z, Salamat N, Ayub M, Ghani E. Anti-hepatits
B core antigen testing, viral markers, and occult hepatitis B
virus infection in Pakistani blood donors: implications for
transfusion practice. Transfusion. 2007;47:74-9.
13. Phikulsod S, Oota S, Tirawatnapong T. Working Group for NAT
Studyin Thai Blood Donations: One year experience of nucleic
acid technology testing for human immunodeficiency virus
type 1, hepatitis C virus and hepatitis B virus in Thai blood
donations. Transfusion. 2009;49:1126-35.
14. Jutavijittum P, Andernach IE, Yousukh A, Samountry B,
Samountry K, Thammavong T, et al. Occult hepatitis B infections
among blood donors in Lao PDR. Vox Sang. 2014;106:31-7.
15. Kleinman SH, Kuhns MC, Todd DS. Retrovirus Epidemiology
DonorS: Frequency of HBV DNA detection in US blood donors
testing positive for the presence of anti-HBc: implications for
transfusion transmission and donor screening. Transfusion.
2003;43:696-704.
16. Bhattacharya P, Chandra PK, Datta S, Banerjee A, Chakraborty
S, Rajendran K, et al. Significant increase in HBV, HCV, HIV and
syphilis infections among blood donors in West Bengal, Eastern
India 2004-2005: exploratory screening reveals high frequency
of occult HBV infection.World J Gastroenterol. 2007;13:3730-3.
17. Chaudhuri V, Nanu A, Panda SK, Chand P. Evaluation of
serologic screening of blood donors in India reveals a lack of
correlation between anti-HBc titer and PCR-amplified HBV
DNA. Transfusion. 2003;43:1442-8.
18. Dhawan HK, Marwaha N, Sharma RR, Chawla Y, Thakral B,
Saluja K, et al. Anti-HBc screening in Indian blood donors: Still
anun resolved issue. World J Gastroenterol. 2008;14:5327-30.
19. Doda V, Arora S, Kirtania T. Serological characterization of
occult hepatitis B virus infection among blood donors in India.
Transfus Apher Sci. 2014. p. 30.
20. Chamni N, Louisirirotchanakul S, Oota S, Sakuldamrongpanish
T, Saldanha J, Chongkolwatana V, et al. Genetic characterization
and genotyping of hepatitis B virus (HBV) isolates from donors
with an occult HBV infection. Vox Sanguinis. 2014. 17. doi:
10.1111/vox.12178. [Epub ahead of print].
21. Shyamala V. Factors in enhancing blood safety by nucleic acid
technology testing for human immunodeficiency virus, hepatitis
C virus and hepatitis B virus. Asian J Transfus Sci. 2014;8:13-8.
22. Nishikawa H, Osaki Y. Clinical Significance of Occult Hepatitis
B Infection in Progression of Liver Disease and Carcinogenesis.
Journal of Cancer. 2013;4:473-80.
23. Seed CR, Kiely P. A method for estimating the residual risk of
transfusion-transmitted HBV infection associated with occult
hepatitis B virus infection in a donor population without
universal anti-HBc screening. Vox Sang. 2013;105:290-8.
24. Taira R, Satake M, Momose S, et al. Residual risk of transfusion-
transmitted hepatitis B virus (HBV) infection caused by blood
components derived from donors with occult HBV infection in
Japan. Transfusion. 2013;53:1393-1404.
25. Lui CJ, Lo SC, Kao JH, et al. Transmission of occult hepatitis B
virus by transfusion to adult and pediatric recipients in Taiwan.
J Hepatol. 2006;44:39-46.
 Chapter 64  Occult Hbv Infection: A Risk for Transfusion  235
26. Levicnik-Stezinan S, Rahne-Potokar U, Candotti D. Anti-HBs
positiveoccult hepatitis B virus carrier blood infectious in two
transfusion recipients. J Hepatol. 2008;48:1022-5.
27. Wang JT, Lee CZ, Chen PJ. Transfusion-transmitted HBV
infection in anendemic area: the necessity of more sensitive
screening for HBV carriers. Transfusion. 2002;42:1592-7.
28. Satake M, Taira R, Yugi H. Infectivity of blood components
with low hepatitis B virus DNA levels identified in a look back
program. Transfusion. 2007;47:1197-1205.
29. Tani Y, Aso H, Matsukura H, Tadokoro K, Tamori A, Nishiguchi
S, et al. Significant background rates of HBV and HCV
infections inpatients and risks of blood transfusion from
donors with lowanti-HB ctitres or high anti-HB ctitres with
high anti-HB stitres in Japan: a prospective, individual NAT
study oftransfusion- transmitted HBV, HCV and HIV infections.
Vox Sang. 2012;102:285-93.
30. Sabat J, Dwibedi B, Dash L, Kar SK. Occult HBV Infection in
Multi Transfused Thallasemic Patients. Indian J Pediatr. 2014.
p. 15.
31. Allain JP. International collaborative studyproposal for
the characterization of occult hepatitis B virus infection
identified by nucleic acid or anti-HBc screening. Vox Sang.
2007;92:254-7.
32. Satoh K, Iwata-Takakura A, Yoshikawa A, Gotanda Y, Tanaka T,
Yamaguch T, et al. A new method of concentrating hepatitis B
virus (HBV) DNAand HBV surface antigen: an application of
the method to the detection of occult HBV infection. Vox Sang.
2008;95:174-80.
33. Vermeulen M, Dickens C, Lelie N. Hepatitis B virustrans
mission by blood transfusion during 4 years of individual
donationnucleic acid testing in South Africa: estimated and
observed window period risk. Transfusion. 2012;52:880-92.
Current Status of Bacterial
Detection in Blood Components
Mina Sidhu
Abstract
Transfusion of allogenic blood and its components are
a potential source of infection by a variety of known and
unknown transmissible agents. Blood safety and prevention
of transfusion transmitted pathogens are the principal aim
of transfusion medicine. To improve the Blood Safety many
improvements have been made in Donor selection process,
Implementation of new technologies like third and fourth
generation ELISA and nucleic acid amplification techniques
(NAT) in detection of transfusion transmitted viral infections.
Bacterial contamination of blood components and prevention
of transfusion transmitted bacterial infections (TTBIs) still
remains the major challenge in transfusion medicine. Platelet
concentrates (PCs) represent one of the highest risks for
bacterial infection due to storage at room temperature with
continuous agitation. Rate of bacterial contamination of PCs
is 1in 1000–3000. In general, bacterial screening of blood
components is done by conventional or automated culture
methods. Many developments have been made in the rapid
direct and indirect method of bacterial detection mainly for
PCs including NAT, pan genera detection (PGD). Fluorescent
activated cell sorting (FACS), noninvasive O2 detection, etc. for
bacterial detection. The ideal system for detection of bacterial
contamination should be highly sensitive and specific, should
be able to generate fast results, detect low levels (as low as 1
CFU/blood bag) of bacterial load and affordable. Currently
no such system is available. Nevertheless various pathogen
reduction techniques are available for PCs and plasma.
Therefore, automated culture systems can be complemented
with rapid systems for detection of TTBIs.
Key words: Transfusion transmitted bacterial contamination,
Platelet concentrate, Culture systems, NAT, PGD, FACS.
Introduction
Transfusion of allogenic blood and its components are
a potential source of infection by a variety of known and
65
unknown transmissible agents. Blood safety and prevention
of transfusion transmitted pathogens are the principal aim
of transfusion medicine. To improve the blood safety many
improvements have been made in Donor selection process,
Implementation of new technologies like third and fourth
generation ELISA and nucleic acid amplification techniques
(NAT) in detection of transfusion transmitted viral infections,
reducing the risk to the minimum, thus increasing the quality
and safety of the Blood Component to the highest level ever.1
As a result of this success, transfusion transmitted bacterial
infections (TTBI) have emerged as greatest threat to blood
safety and is an important transfusion related adverse event.
In United States, bacterial contamination is considered the
second most common cause of death overall from transfusion
(after clerical errors) with mortality rate from bacterial
sepsis ranging from 1:20,000 to 1:85,000. Estimates of severe
morbidity and mortality range from 100 to 150 transfused
individuals each year.2
Platelet concentrates (PCs) represent one of the highest
risks for bacterial infection. This is due to the required
storage conditions for PCs in gas-permeable containers at
room temperature 22oc to 24oc with constant agitation to
maintain the function and survival, which support bacterial
proliferation from low contamination levels to high titers.3
The risk of bacterial contamination of platelet units is 1 in
1000 to 3000, 50–250 times higher than the combined risk
of human immunodeficiency virus, hepatitis B and C virus
and human T-cell leukemia virus 1 and 2, clinical sepsis in
about 1/20,000 and related fatality in 1/60,000 transfusion.4-6
The common pathogens isolated in platelet concentrate are
mainly skin flora, including Staphylococcus epidermidis and
Propionibacterium acnes.
Sepsis associated with the transfusion of red cell blood
component is a rare event because of storage at 2–6oC,
where bacteria generally do not grow. From 1976 through
September 1998, 26 fatalities were reported to US food and
drug administration. Most common organism involved was
Yersinia enterocolitica.3 Coagulase positive Staphylococcus,
 Chapter 65  Current Status of Bacterial Detection in Blood Components  237
gram-negative Serratia liquefaciens and Prionibacterium spp.
are also isolated from the whole blood and red cells culture.
Varied incidence ranging from 2–4 per 1000 units4, 1 per
70807 and 1 per 31,3858 are recorded.
Plasma and cryoprecipitate stored frozen and thus are
rarely associated with contamination. However, in some
cases Pseudomonas cepacia and Pseudomonas aeruginosa
have been cultured from cryoprecipitate and plasma thawed
in contaminated water.1
Clinical Relevance
The unique aspect of most bacterial species when compared
to viral pathogens is that they can really proliferate in the
nutrient-rich blood product environment during storage.
This is particularly true in platelet products that are stored
at room temperature, rather than in the cold. It is estimated
that the level of contamination at the time of collection is
generally relatively low, probably on the order of 1–10 colony
forming units/mL or less. Once the product is contaminated,
the inoculated bacterial seed can proliferate within hours
to significant level. Such quantities of bacteria infused with
the transfusion product over a short period of time can
cause bacteremia that may progress to sepsis and death. The
outcome of a contaminated transfusion is highly dependent
on the amount of bacteria transfused, the type of bacteria and
its pathogenicity for humans, the rate of transfusion, and the
clinical status of the recipient. Immunosuppressed patients
and older individuals with poor nutritional status will be the
most susceptible population.2
Thus, there is significant concern about the demand for a
“Safe or Safer” blood supply in order to provide the best and
safest patient care. This risk has become “unacceptable” in
a climate where the blood supply remains “the safest than it
has ever been.2 American Association of Blood Banks (AABB)
standard 5.15.1 in 2004 recommended the implementation of
methods to limit or detect bacterial contamination.
Methods to reduce Risk of post
transfusion sepsis
There are multiple strategies to reduce transfusion related
bacterial infection, these include better donor selection to
defer the donors with risk of bacteremia, proper phlebotomy
site disinfection and diversion of first 10–30 mL of whole
blood in predonation sampling bag. These strategies have
significantly contributed to reduce the contamination of
whole blood, Red cells and platelet concentrate by the skin
bacterial flora. The suspicion that increased transfusion
transmitted bacterial infections may be due to long shelf life
to reduction in shelf life to 3 days in Japan, 4 days in Germany
and 5 days in USA.3 Microbiological monitoring of blood
components is essential to detect TTBIs.
Methods to detect bacterial
contamination in blood component
The ideal test for detection of bacteria in blood components
should be highly sensitive and specific in order to detect low
number of bacteria, results should be generated fast as the
shelf life of platelets is short, simple and affordable.9
Several tests that are routinely used to check the quality
control are based on metabolic changes, morphology,
microscopy and culture.
The detection methods can be grouped into: (1) Indirect
rapid bacterial detection test, (2) Direct detection methods,
(3) Direct rapid detection methods.
Indirect Rapid Bacterial
Detection Tests
pH
When bacteria grow in PCs they consume glucose and
produce acid. Production of acid is accompanied by a
decrease of pH, which can be measured by a pH meter or a
dipstick. Also the decrease in glucose can be measured with
an automated glucose analyzer or a dipstick. During bacterial
growth, the decrease of pH leads to a morphological change of
the platelets. The morphological alternation is thought to be
predictive for loss of viability of platelets. Swirling is an effect
related to the discoid morphology of platelets in movement.
This scattering is reduced or absent in non discoid platelets,
used to detect bacterial growth. Although, the methods
mentioned above are inexpensive, simple to perform, fast
and the sensitivity is very low. Bacterial loads as high as 107
to 108 CFU/mL can be detected.10 Many new developments
made in indirect methods are described below.
Noninvasive pH Measurement
This sensor comprises a pH-sensitive fluorescent membrane
fixed to a clear window inside a small sensor tube, which is
welded in the rim of the PC container. Fluorescence of the
sensor membrane is measured through the window using
a fiberoptic probe and the BCSI pH SAFE reader calculates
pH based on the ratio of yellow and red fluorescence
measured.11,12 Due to the lack of sensitivity, these methods
should be discontinued in favor of a more sensitive detection
method.13
Noninvasive Oxygen Measurement
A new continuous noninvasive bacterial detection method
has been developed using O2 measurements in the platelet
fluids. The principle of the sensor operation is based on the
quenching of luminescence caused by collision between
238  Section 3  Blood Safety
molecular oxygen and luminescent dye molecules in the
excited state. Oxygen concentration in platelet fluids is
continuously detected by oxygen sensing probes and
registered every 20s. Probes can be labeled by barcodes and
integrated into the platelet storage bag. On the basis of the
generated baseline data for sterile PCs, a bacterial-positive
blood product was defined as showing an O2 saturation of
<10% because all negative samples had oxygen saturation
higher than 30% over a time period of 7 days. A major
benefit of the described PreSense O2 system is a continuous
measurement in a closed system.14
Platelet Aggregation
Störmer et al.15 evaluated the suitability of platelet aggregation
as a parameter for bacterial contamination of apheresis PCs in
comparison to bacterial count calculation. They demonstrated
that platelet aggregation was reduced in bacterially
contaminated products in comparison to sterile products. The
level of reduction was dependent on donors and the growth
properties of the bacterial strain in the product.
Direct Methods of Bacterial
detection
Microscopy
Although a higher sensitivity is obtained from microscopic
examination with either gram stain or acridine orange, the
sensitivity remains poor. The lower detection limits are 105 to
106 CFU/mL for gram stain and 104 to 105 CFU/mL for acridine
orange stain.16
Scansystem
The scan system (Hemosystem, Marseilles, France) is
approved by the FDA for quality control of platelets. This
method detects bacteria using a laser-based, solid phase
scanning cytometry detection method. Platelet samples are
incubated with monoclonal antibodies that aggregate the
platelets in the sample, which are then removed by filtration
step that allows the passage of bacteria. It is then mixed with
fluorescent labeling agent picogreen. By vacuum pump
bacteria is retained on the black membrane. The membrane
is scanned with laser and bacteria are counted. The system
is rapid and has sensitivity of 50 CFU/mL.17,18 This system is
no longer available in the market due to complex analysis
procedure and it also does not differentiate between dead
and live bacteria.3
Culturing Methods
Many countries have implemented automated culture
systems using the negative-to-date concept to screen PCs
for bacterial contamination.19-21 Cultivation or incubation
methods require time for signal production as they depend
on microbial growth. Therefore, samples are collected from
the PCs early in the shelf life, 24 h after donation. 4–10 mL
samples are then inoculated into aerobic (and anaerobic)
culture bottles and incubated in the automated culture system
up to 7 days at 35–37 °C.22 PCs with a negative diagnostic
status are released but cultivation is continued. If the result
status changes from negative to reactive, physicians have to
be informed immediately and products need to be recalled.
If PCs were already transfused a look-back procedure has to
be initiated.23 Three automated culture methods have been in
routine use or have been validated for platelet quality control:
the BacT/ALERT system (BioMérieux, France), the Bactec
(BD Diagnostics, USA), and the VersaTrek system (Trek
Diagnostics, USA).
The BacT/Alert is by far the most common in reports and
has been cleared for this application by the Food and Drug
Administration (FDA).23,24 It monitors CO2 production due to
bacterial growth, which changes the color of a gas-permeable
sensor at the bottom of the culture bottle from grey to yellow.23
The Bactec system is very similar to the BacT/Alert system
but uses a fluorimetric instead of the colorimetric detection
principle.24 The VersaTREK monitors bacterial growth by
detecting pressure changes in the headspace of the blood
culture bottle secondary to gas consumption/production.25
BacT/Alert
The BacT/Alert is by far the most commonly used and it
monitors CO2 production due to bacterial growth, which
changes the color of a gas-permeable sensor at the bottom
of the culture bottle from grey to yellow.19-21,23 A report of
“negative” should not be interpreted as meaning that the
original product is sterile. The negative status could be due
to under inoculation of the bottle, no organisms present in
the inoculum, the number of organisms were too small for
detection, or a culture bottle/medium that does not support
the growth of the organism.26,27
The BacT/Alert system is very sensitive and reliably detects
contamination of platelets inoculated to 5–10 CFU/mL.1
Platelets are released negative to date result which means
that on the day of release the culture bottles of the platelets
are negative for bacterial growth.19,20
Enhanced Bacterial Detection System
Another principle that was introduced into routine practice
and cleared by the FDA is the Pall enhanced bacterial
detection system (eBDS) system (Pall Corporation, USA),
now Hemonetics eBDS (Hemonetics Corporation, USA). This
method is a novel culture-based system which assesses the
reduction of the O2 content in the head space of a sample
pouch containing an enriched medium, compared to
 Chapter 65  Current Status of Bacterial Detection in Blood Components  239
ambient O2, as a marker for bacterial growth. The method
involves docking an eBDS sample pouch onto the tubing of
platelet bag, transfer of an aliquot of 2–3 mL of the platelet
product into the sample pouch, which is then separated from
the platelet unit using a heat sealer. The eBDS pouch is then
incubated at 35 oC for 24 hours with continues agitation.
Pouches are then removed from the incubator, kept at
room temperature for at least 2 minutes and the probe of
the oxygen analyzer inserted into the sample port of the
pouch. The oxygen analyzer automatically reads the oxygen
concentration in the sample pouch. Bacterial contamination
is assumed if the O2 reading is less than 12.5%, which is the
cut-off value between a positive and negative reading.28,29
Using this technology only aerobes and facultative anerobes
are detected. The sensitivity of the Pall eBDS system is similar
to the BacT/ALERT system in the order of 1–10 CFU/mL.29
Microcalorimetry
Measuring heat due to replicating microorganisms in
culture has been used for detection of bacteria in PCs. A
microcalorimetry thermostat is used to measure the heat
flow curves. Any heat generated or absorbed by the sample is
measured continuously over time. Trampuz et al.30 artificially
contaminated PC samples with different bacterial species at
cell counts of 1–105 CFU/mL.
Direct Rapid Detection Methods
Several detection methods suitable for use at the time of issue
of platelet products are currently developed and include the
following:
Pan Genera Detection Test System
The platelet pan genera detection (PGD) test system, (Verax
Biomedical, Worcester, MA), a rapid detection system. The
PGD test system detects the presence of conserved antigens
on the surface of bacteria, lipoteichoic acid (LTA) found on
gram-positive bacteria and lipopolysaccharide (LPS) found
on gram-negative bacteria. Lipoteichoic acid and LPS are
primary constituents of the cell walls of bacteria and are
detected by the use of specific antibodies. The PGD test
system is a rapid test in which nonautomated procedures are
involved and results are given quickly. Analytical sensitivity
of the test was determined for 10 bacterial strains and ranged
from 103 to 105 CFU/mL. The test is cleared by FDA only to be
used as a supplement quality control test.31 Because of its low
sensitivity, the test cannot be used for screening PCs.
Fluorescence Activated Cell Sorting Analysis
For fluorescence activated cell sorting (FACS) analysis,
bacteria are stained for 5 minutes with the membrane
permeable dye thiazole orange, which stains the nucleic
acids of viable and dead cells. The bacteria are detected
by flow cytometric analysis. The sensitivity of the assay is
a approximately 105 CFU/mL, but a preincubation step
in a bacterial growth medium increases the sensitivity
considerably to 10 CFU/mL. Bacterial detection with FACS
analysis without the preincubation step takes approximately
20 minutes. Not all bacteria are easily detected by FACS
analysis. Sometimes due of the influence of the background
signal, the quantification of the bacterium is difficult.32
The BactiFlow assay detects and enumerates bacteria
by fluorescent labeling of viable cells. A nonfluorescent
fluorochrome passes the cell membrane of cells with intact
membrane integrity and enzymatic activity, and is cleaved
by intracellular esterases. To reduce the background noise,
the esterase activity of platelets is selectively eliminated by
enzymatic digestion. The analytical sensitivity of this assay is
validated to be 300–500 CFU/mL.33
Nucliec Acid Amplification Assay/Techniques
Nucliec acid amplification techniques (NAT) assays are widely
used in blood banks to screen for transfusion transmitted
infections. Bacterial DNA is detected by Real time PCR. Two
generic assays have been well defined.34,35 These are based
on 16S rDNA34 and 23S rDNA35 gene that is conserved among
bacteria and is present in multiple copies within the genome.
Because of this it is possible to detect all bacteria that are
relevant for TTBI with single PCR. Analytical sensitivity vary
from 5–50 CFU/mL. False positive results could be caused
by reagent contamination or a cross-reaction with human
nucleic acids. Only the assay developed by Sireis et al.36
received CE-IVD (in vitro diagnostics) certification and is
commercially available.
Table 1 compares the various properties of the available
detection methods.9
Bacterial Peptidoglycan Chromogenic
Immunoassay
This method is being developed as BacTx by Immunetics,
IUnc, Boston, MA and consists of a test kit containing all
needed reagents and a chromogenic reader. The system has
a turnaround time of less than one hour. The system detects
bacterial peptidoglycan in gram-positive and gram-negative
bacteria when a peptidoglycan binding protein triggers
enzymatic conversion of chromogenic substrate to a visible
product with measurable absorbance.37 The BacTx received
FDA clearance for bacterial contamination of PCs in 2012.3
Pathogen Reduction
Along with methods that detect bacteria, there are methods
that reduce the amount of bacteria in PCs. An advantage of
240  Section 3  Blood Safety

Table 1: Properties of tests available for detection of bacteria in platelet concentrates

Detection system Sensitivity Testing time PC input Relative cost Labor
(CFU/mL) (mL) of reagent
Metabolic parameters/morphology 107 to 108 <5 minutes <1 Low Low
Gram/acridine orange stain 105 to 106 30 minutes <0.1 Low High
Optimized Scan system 50 CFU/ mL 25 hours 3 Medium High
BacT/Alert 1–10 CFU/mL 20 hours–7 days 5–10 Medium Medium
eBDS PALL system 1–10 CFU/mL 24–25 hours 2–3 Medium Medium
PGD Test system 103 to 105 30–70 minutes 0.5 High Medium
FACS analysis 10–105 6.5 hours 3 Low Medium
NAT assay 22–50 CFU/mL 2.5–4 hours 0.2–2.4 High High
Microcalorimetry 1–105 8–73 hours 1 Low Medium

pathogen reduction is that it inactivates a broad range of
pathogens such as bacteria, viruses, parasites and fungi.
There are several approaches for pathogen reduction of PCs.
Intercept (Fenwall, formerly Baxter Corporation), Mirasol
(Gambro BCT), and the Theraflex UV pathogen reduction
system (Macopharma). The intercept method uses psoralen
combined with long wave length UV light to inactivate a broad
range of contaminating pathogens, including bacteria. The
Mirasol technique uses riboflavin (Vitamin B) in combination
with UV to reduce pathogens in blood products.38 Although
attractive, pathogen reduction of PCs is costly.
important milestone in improving bacterial safety of blood
components. Due to delay in generation of results and
problem of false positive cases, newer rapid techniques like
BactiFlow, PGD and NAT testing can be adopted. The future
detection of TTBI lies in the tests that are rapid, sensitive and
are able to detect broad range of bacteria. Undoubtly, NAT
is fast detection system and can be used close to the time
of transfusion. Very low level of bacteria can be detected.
However, cost effectiveness study should be done to see if
implementation of NAT assay is affordable, for the detection
of Transfusion transmitted bacterial infections.

Technologies Under Development References

Wang et al.39
developed a strategy for detecting specific
nucleic acid fragments by combining universal PCR with
lateral-flow dipstick (LFD) technology. This assay utilizes
the amplification of genes followed by hybridization by
a set of probes that are specific for the conserved region of
the target. Colloidal gold-labeled avidin and specific anti-
FITC (fluorescein isothiocyanate) antibodies immobilized
on the LFD nitrocellulose membrane then capture a probe-
amplicon complex, producing a visible red line on the
membrane. According to the results of the group, the total
analysis time of LFD technology is only about 2 hours, but the
sensitivity varies depending on the bacterial species from 5 to
104 CFU/mL.
A spore-based biosensor for detecting bacteria in real
time has been recently developed by Rotman and Cote.40
The system, termed LEXSAS, exploits the ability of spores to
produce fluorescence when sensing neighboring bacterial
cells. The LEXSAS contains microbial spores suspended in
an enzymatic substrate that produces a germinant upon
enzymatic catalysis combined with diacetate fluorescein, a
component that fluoresces when hydrolyzed by esterases.
Signal to noise ratio values are determined by comparing
signals from contaminated units to signals from the control.
To conclude, culture methods are gold standard in
bacterial detection. Automation in culture methods is
1. Brecher ME, Hay NS. Bacterial Contamination of Blood
Components. Clin Micribiol Rev. 2005;18(1):195-204.
2. Hillyer DC, Josephen DC, Blajchman MA, G Jaroslav, Epstein SJ,
Goodman LJ. Bacterial contamination of blood components;
Risks, Strategies and Regulation. Joint ASH and AABB
Educational session in transfusion medicine. Hematology
2003;1:573-89.
3. Stormer MA, Vollmer TB. Diagnostic methods for platelet
bacteria screening. Transfus Med Hemother. 2014;41:19-27.
4. Blajchman MA. Incidence and Significance Bacterial
contamination of blood products. Dev Biol. 2002;108:59-67.
5. Blajchman MA, M Goldman. Blood product associated
Bacterial sepsis. Trans Med Rev. 1991;5:73-83.
6. Blajchman MA, M Goldman. Bacterial contamination of Platelet
concentrates: Incidence, significance and preventation. Semi
Hemat. 2001;38:4.
7. Barret BB, Anderson JW, Anderson KC. Srategies to reduce
bacterial contamination of blood components. Transfusion.
1993;133:228-33.
8. Dzieczkowki JS, Barret BB, Nester D, et al. Characterization
of reactions after exclusive transfusion of white cell reduced
blood components. Transfusion. 1995;35:20-5.
9. Rood IGH, Petterson A, Korte D de, et al. Reducing the risk
of transfusion transmitted bacterial infectionsin platelet
concentrate. CE Update: LabMedicine. 2008;39:553-8.
10. Wagner SJ, Robinette D, Evaluation of swirling, pH and glucose
test for the detection of bacterial contamination in platelet
concentrate. Transfusion. 1996;36:989-93.
 Chapter 65  Current Status of Bacterial Detection in Blood Components  241
11. Barker LM, Nanassy OZ, Reed MW, et al. Multiple pH measure-
ment during storage may detect bacterially contaminated plate-
let concentrates. Transfusion. 2010;50:2731-37.
12. Gkoumassi E, Klein-Bosgoed C, Dijkstra-Tiekstra MJ, et al.
Noninvasive pH monitoring of platelet concentrates: A large
field test. Transfusion. 2013;53:2287-92.
13. Yomohow R, Brecher ME. pH and Glucose test of SDP should
be discontinued in favour of more sensitive detection method.
Transfusion. 2005;45:646-8.
14. Mueller MM, Hourfar MK, Huber E, et al. Oxygen measure­
ments in platelet fluids—a new noninvasive method to
detect bacterial contaminations in platelets. Transfus Med.
2012;22:211-6.
15. Störmer M, Petrescu-Jipa VM, Radojska S, et al. Measuring
platelet aggregation may detect bacterial contamination
in platelet concentrates. 23rd Regional Congress of the
International Society of Blood Transfusion, 2-5 June 2013,
Amsterdam, The Netherlands. Vox Sang. 2013;105(suppl 1):34.
16. Mitchel KM, Brecher ME. Approaches the detection of bacterial
contamination in cellular blood products. Trans Med Rev.
1999;13:132-44.
17. Schmidt M, Weis C, Hect J, et al. Optimized Scansystem platelet
kit with enhanced sensitivity. Detection within 24 hours after
spiking. Vox Sang. 2005;89:135-9.
18. Ribalt S, Harper K, Grave L, et al. Rapid screening method for
detection of bacteria in platelet concentrate. J Clini Microbiol.
2004;42:1903-8.
19. Störmer M, Petrescu-Jipa VM, Radojska S, et al. Measuring
platelet aggregation may detect bacterial contamination
in platelet concentrates. 23rd Regional Congress of the
International Society of Blood Transfusion, 2-5 June 2013,
Amsterdam, The Netherlands. Vox Sang. 2013;105(suppl 1):34.
20. Eder AF, Kennedy JM, Dy BA, et al. Bacterial screening of
apheresis platelets and the residual risk of septic transfusion
reactions: The American Red Cross experience (2004-2006).
Transfusion. 2007;47:1134-42.
21. Koopman MM, van’t Ende E, Lieshout-Krikke R, et al. Bacterial
screening of platelet concentrates: Results of 2 years active
surveillance of transfused positive cultured units released as
negative to date. Vox Sang. 2009;97:355-7.
22. European Pharmacopeia Update: Microbiological control of
cellular products. Chapter 2.6.27.
23. Schmidt M, Sireis W, Seifried E. Implementation of bacterial
detection methods into blood donor screening-overview of dif-
ferent technologies. Transfus Med Hemother. 2011;38:259-65.
24. Murphy WG, Coakley P. Testing platelet components for
bacterial contamination. Transfus Apher Sci. 2011;45:69-74.
25. Nanua S, Weber C, Isgriggs L, et al. Performance evaluation of
the Versa TREK blood culture system for quality control testing
of platelet units. J Clin Microbiol. 2009;47:817.
26. Benjamin RJ, Wagner SJ. The residual risk of sepsis: Modelling
the effect of concentration on bacterial detection in two bottle
culture system and estimation of false nagatve culture rates.
Transfusion. 2007;47:1381-9.
27. Boekhorst PA, Beckers EA, Vos MC, et al. Clinical significance of
bacteriological screening in platelet concentrate. Transfusion.
2005;45:514-9.
28. Pietersz RN, Engelfret CP, Persin KHW. Detection of bacterial
contamination in platelet concentrate. Vox Sang. 2007;93:
260-7.
29. Fournier Wirth C, Deschaseaux M, Defer C. Evaluation
of enhanced Bacterial Detection System for screening of
contaminated platelets. Transfusion. 2006;46:220-4.
30. Trampuz A, Salzmann S, Antheaume J, et al. Micro­
calorimetry: A novel method for detection of microbial
contamination in platelet products. Transfusion. 2007;47:
1643-50.
31. Vollmer T, Hinse D, Kleesiek K, et al. The Pan Genera detection
immunoassay: A novel point-of-issue method for detection
of bacterial contamination in platelet concentrates. J Clin
Microbiol. 2010;48:3475-81.
32. Dreier J, Vollmer T, Kleesiek K. Novel flow cytometry-based
screening for bacterial contamination of donor platelet
preparations compared with other rapid screening methods.
Clin Chem. 2009;55:1492-1502.
33. Vollmer T, Dreier J, Schottstedt V, et al. Detection of bacterial
contamination in platelet concentrates by a sensitive flow
cytometric assay (BactiFlow): A multicentre validation study.
Transfus Med. 2012;22:262-71.
34. Mohammadi T, Pietersz RN, Vandenbroucke-Grarib CM, et al.
Detection of bacteria in platelet concentrate: Comparison of
broad range real time 16S rDNA polymerase chain reaction
and automated culturing. Transfusion. 2005;45:731-6.
35. Stormer M, Kleesiek K, Dreier J. High volume extraction of
nucleic acid by magnetic bead technology for ultrasensitive
detection of bacteria in blood components. Clin Chem.
2007;53:104-10.
36. Sireis W, Ruster B, Daiss C, et al. Extension of platelet shelf life
from 4 to 5 days by implementation of a new screening strategy
in Germany. Vox Sang. 2011;101:191-9.
37. Jacobs MR, Bajaksouzian S, Yomtovian R, et al. Detection
of bacteria in Leukocyte-reduced whole blood derived
platelet units using the Immunetics BacTx Test. Transfusion.
2010;50:194A.
38. Pelletier JP, Transue S, Snyder EL. Pathgen inactivation
techniques. Best Pract Res Clin Hematol. 2006;19:205-42.
39. Wang J, Wang X, Li Y, et al. A novel, universal and sensitive
lateral-flow based method for the detection of multiple
bacterial contaminations in platelet concentrations. Anal Sci.
2012;28:237-41.
40. Rotman B, Cote MA. Application of a real-time biosensor to
detect bacteria in platelet concentrates. Biochem Biophys Res
Commun. 2003;300:197-200.
Pathogen Reduction Technology
Kanchan Bhardwaj
Pathogen reduction technology is one more positive step
towards blood safety designed to reduce the risks from bacteria,
viruses and parasites in donated blood components. This
system proactively and safely reduces the risk of transfusion—
transmitted diseases by inactivating susceptible pathogens
and residual leucocytes that may be present in donated
blood components. This offers the potential to inactivate
untested for and emerging pathogens before they become
a major transfusion risk to patients. While not all pathogens
are susceptible to inactivation, this system offers the most
comprehensive approach available. The development and
adoption of pathogen reduction technology (PRT) continues.
The ability to reduce transmission of a broad range of infectious
and noninfectious threats remains the primary reason for
PRT.1 The robust inactivation technology that is compatible
with blood compo­ nent processing procedures, offers the
potential for significantly improving transfusion safety. To
be highly effective, a successful technology must inactivate
pathogens in extracellular, intracellular, and nuclear
compartments including pathogen nucleic acid sequences
integrated into donor leucocytes. Technology capable of
inactivating residual leucocytes may confer additional benefits
owing to inhibition of critical leucocyte functions, including
cytokine synthesis, lymphocyte proliferation and antigen
presentation. Nucleic acid targeted pathogen inactivation
process offers the potential to inactivate leucocytes as well as
infectious pathogens.
Pathogen inactivation in Platelet
Concentrates
The pathogen inactivation in platelet concentrates is
divided into two basic groups: nucleic acid targeted and
photodynamic methods. These utilize photochemical
reactions in an ex vivo treatment combining a photo reactive
compound and ultraviolet light. Nucleic acid targeting using
a novel psoralen amotosalen has undergone clinical trials
and been commercialized. These methods relay on nucleic
66
acid—specific adduct formation as a primary mechanism
for pathogen inactivation. Psoralen is furocoumarins,
which reversibly intercalate into helical regions of DNA and
RNA. Under illumination with UVA, psoralens react with
pyrimidine bases to form covalent adducts and cross links
with nucleic acids. Bacteria, protozoa, viruses and nucleated
cells modified with psoralens are unable to replicate.2-6
Photodynamic method utilizes Riboflavin which produces
active oxygen species which oxidizes guanine in nucleic
acid and prevents replication of the pathogen genome in
addition to nucleic acid reactions as a primary mechanism
for pathogen inactivation.
Platelet concentrates with
Amotosalen photochemical
treatment
Helinx technology prevents replication of DNA and RNA
in pathogens without affecting blood components being
treated. Closed system with pooled buffy coat or single
donor platelet concentrate, suspended in approximately 35%
plasma and 65% platelet additive solution, is connected to a
container of amostosalen through which it is passed. This is
exposed to UVA for 3 minutes after which it is transferred to a
compound absorption device (CAD) for 4 hours to passively
reduce the residual amotosalen to low levels (<0.5 µmol/L)
after which the platelets are transferred to a final container for
storage for 5–7 days under conventional storage conditions.
The Intercept blood system developed by Cerus is the first
pathogen inactivating system approved to treat platelets for
transfusion. It has been approved for use in Europe. Safety
profile of treated platelets is similar to that of conventional
platelets.7-9 Hemovigilance reporting system to examine the
incidence of acute reactions in the 24 hours post-transfusion
period confirmed a low rate of acute transfusion reaction
and tolerability of the treated platelets.10 Impact of intercept-
treated platelets based on proteomic analyses have been
reported, and the overall interpretation of proteomic findings

suggests some minor changes although, there is no evidence
for a potential clinical concern for transfusion recipients.11 It
is also being developed for treatment of plasma and red cells.
Intercept-treated platelets are in routine use in 21 countries
and plasma in routine use in 12 countries.1
Riboflavin and Ultraviolet light
treated Platelets
This method involves the suspension of platelets in 100%
donor plasma. It acts against enveloped viruses, nonenve­
loped viruses, protozoa and some bacteria. It reduces the
infectious pathogen load by 99.9% in > 70% of the pathogens
to the limit of detection. It causes 6 log reduction of
leucoreduction hence preventing CMV transmission. In this
the platelets are transferred into an illuminating bag to which
riboflavin is added. This is illuminated in an illuminator for
4–10 minutes after which it is ready for storage or transfusion.
Platelet additive solution may be needed to be added after
illumination to platelet concentrates with reduced plasma
content to ensure adequate storage conditions for up to 7
days. Functional adequacy of the platelets was maintained.
Terumo BCT Mirasol PRT is based on this principle (CE
approved). This is also used for plasma before freezing
it. Recent clinical research on Mirasol-treated platelets
and plasma PRT has focused on studies that quantify the
performance of white blood cell inactivation, with strong
evidence of advantages over gamma irradiation.12 Mirasol-
treated platelets are in routine use in 18 countries and plasma
in routine use in 11 countries.
Methylene blue treated Platelets
Initial success in developing a novel procedure that uses
short-wave ultraviolet light (UVC 200–280 nm) without
addition of a photoactive chemical to treat platelets has
been reported and is currently being evaluated for safety and
efficacy.13,14 Published information suggests both the UVC
platelets treatment intended for apheresis and buffy-coated
derived platelets and the more widely available methylene
blue (MB) treatment for plasma from apheresis and whole
blood derived plasma are effective at inactivation of many
parasites, bacteria and viruses.15,16
Pathogen Inactivation in Fresh Frozen
Plasma Preparation
Solvent detergent (SD) process for inactivation of enveloped
viruses in plasma is used. It is not effective against non-
enveloped viruses. It is used for factor replacement in
congenital and acquired factor deficiencies. As it does not
contain von Willebrand factor (vWF), it has been advocated
in therapeutic plasma exchange (TPE) therapy of thrombotic
thrombocytopenic purpura (TTP). There is mild to marked
decrease of antithrombotic proteins: protein S, plasmin
Chapter 66  Pathogen Reduction Technology  243
inhibitor and coagulation factors. It involves pooling of
100–2500 units of plasma and is not amenable to a single unit
viral inactivation process.
Methylene blue (MB) is used for pathogen reduction
for plasma by Macopharma THERAFLEX MB. Based on
hemovigilance data for methylene blue (MB)-treated plasma,
a significantly higher incidence of severe allergic reactions or
other adverse events in comparison with other fresh frozen
plasma types could not be confirmed.17,18 Methylene blue and
long wavelength visible light has been used in clinical practice
for preparation of FFP which utilizes active oxygen species.
It is not effective against intracellular species. Theraflex MB-
treated plasma is in routine use in 15 countries.1
A photochemical method using amotosalen and UVA
light has been developed to inactivate viruses in single units
of plasma prepared as FFP. Riboflavin treated and subjected
to UVA leads to pathogen inactivation in short time (frozen
within 8 hours), removing time constraints associated with
FFP processing.
Potential System for Pathogen
Inactivation in Red Cell concentrates
Nucleic acid—targeted processes that do not require light
activation are two groups of compounds which have been
used for pathogen reduction in red cell concentrates viz.
Inactine (PEN-110) and frangible anchor linker effectors
Frale called S-303. This approach offers the potential to
minimize nonspecific damage by active oxygen species to red
cells and plasma proteins.19 Published research20,21 on whole
blood PRT currently in development provides evidence of
pathogen reduction, white cell inactivation and reduction in
antigen expression by Mirasol PRT.
Conclusion
Both controlled clinical studies and postmarketing surveil­
lance are necessary to further establish the safety and
effectiveness of each PRT method. Recent evidence does not
suggest an obvious safety threat for any of the current PRT
methods in routine use. The relative value in terms of patient
health and health economics, including cost-utility, of these
methods in terms of health gains achieved by PRT remain
insufficiently studied.
References
1. Custer B. Update on pathogen reduction technology. ISBT
Science Series. 2013;8:80-4. Doi10.1111/voxs.12014.
2. Lin L, Dikeman R, Molini B, et al. Photochemical treatment of
platelet concentrates with amotosalen and UVA inactivates a
broad spectrum of bacteria. Transfusion. 2004;44:1496-504.
3. Van Voorhis WC, Barrett LK, Eastman RT, et al. Trypanosoma
cruzi inactivation in human platelet concentrates and plasma
by psoralen and long-wave length UV. Antimicrob Agents
Chemother. 2003;47:475-9.
244  Section 3  Blood Safety
4. Eastman RT, Barrett LK, Dupuis K, et al. Leishmania inactiva­
tion in human pheresis platelets by a psoralen (amotlsalen
HCl) and long-wavelength ultraviolet irradiation. Transfusion.
2005;45:1459-63.
5. Lin L, Hanson CV, Alter HJ, et al. Inactivation of viruses in
platelet concentrates by photochemical treatment with
amotosalen and long-wavelength ultraviolet light. Transfusion.
2005;45:580-90.
6. Grass JA, Hei DJ, Metchette K, et al. Inactivation of leucocytes
in platelet concentrates by psoralen plus UVA. Blood.
1998;91:2180-8.
7. McCullough J, Vesole DH, Benjamin RJ, et al. Therapeutic
efficacy and safety of platelets treated with a photochemical
process for pathogen inactivation: the SPRINT trail. Blood.
2004;104:1534-41.
8. Van Rhenen D, Gullikson H, Cazenave JP, et al. Transfusion
of pooled buffy coat platelets components prepared with
photochemical pathogen inactivation treatment: the euro
SPRITE trail. Blood. 2003;101:2426-33.
9. Snyder E, McCullough J, Slichter SJ, et al. Clinical safety of
platelets photochemically treated with amotosalen HCl and
ultraviolet A light for pathogen inactivation. Transfusion. 2005;
45:1846-75.
10. Osselear JC, Bueno JL, Messe N, Jacquet M, Castro E. Flament
J. Prospective active hemovigilence plan for INTERCEPT
platelets in Europe: status report. Vox Sang. 2005;89:137.
11. Prudent M, Crettaz D, Delobel J, et al. Proteomic analysis of
Intercept-treated platelets. Journal of proteomics. 2012;76
(Spec No.):316-28.
12. Fast LD, DiLeone G, Marschner S. Inactivation of human
white blood cells in platelet products after pathogen reduction
technology treatment in comparison to gamma irradiation.
Transfusion. 2011;51(7):1397-404.
13. Picker SM. Pathogen reduction technologies: the best solution
for safer blood? J Blood Disorders Transf. 2012;3(5):5.
14. Pohler P, Lehmann J, Veneruso V, et al. Evaluation of the
tolerability and immunogenicity of ultraviolet C-irradiated
autologous platelets in a dog model. Transfusion. 2012;52(11):
2414-26.
15. Seghatchian J, Tolksdorf F. Characteristics of the THERAFLEX
UV-Platelets pathogen inactivation system—an update.
Transfus Apher Sci. 2012;46(2):221-9.
16. Steinmann E, Gravemann U, Friesland M, et al. Two pathogen
reduction technologies-methylene blue plus light and
shortwave ultraviolet light-effectively inactivate hepatitis C
virus in blood products. Transfusion. 2012; doi: 10.1111/j.1537-
2995.2012.03858.x. [Epub ahead of print].
17. Bost V, Odent-Malaure H, Chavarin P, et al. A regional
haemovigilance retrospective study of four types of therapeutic
plasma in a ten-year survey period in France. Vox Sang. 2013;
doi: 10.1111/vox.12007. [Epub ahead of print].
18. Seltsam A, Mueller TH. Updated hemovigilance data do not
show an increased risk of allergic reactions to methylene blue-
treated plasma. J Allergy Clin Immunol. 2013;doi:10.1016/j.
jaci.2012.11.046. [Epub ahead of print].
19. Cancelas JA, Dumont LJ, Rugg N, et al. Stored red blood
cell viability is maintained after treatment with a second
generation S-303 pathogen inactivation process. Transfusion.
2011;51(11):2367-76.
20. Reddy HL, Doane SK, Keil SD, et al. Development of a
riboflavin and ultraviolet light-based device to treat whole
blood. Transfusion. 2013;53(Suppl 1):131S-6S.
21. Pidcoke HF, McFaul SJ, Ramasubramanian AK, et al. Primary
hemostatic capacity of whole blood: a comprehensive analysis
of pathogen reduction and refrigeration effects over time.
Transfusion. 2013;53(Suppl 1):137S-49S.
Discarding of Blood and
Blood Components
Bharat Singh, Preeti Dewakar
INTRODUCTION
Blood and blood products are used across the various
departments of a hospital every day to save many lives.
Transfusions are required in accident victims, obstetric
complications, cancer patients, patients with bleeding
disorders, patients undergoing surgeries and premature
babies. Blood banks in India collect about 5 to 5.5 million
units per year, but the total blood requirement is around
9 to 9.5 million units per year. Thus the shortage of blood is
about 40%.1 This mandates special efforts to ensure the safety,
sufficiency and quality of blood and its products which is the
first and foremost responsibility of the Blood Bank and Blood
Transfusion services attached to any hospital.
Shortage of blood supply can be circumvented by either
pumping more resources into the collection of blood and
production of its components or by using the available
resources more efficiently. One way of increasing efficiency is
to reduce wastage and discard rates.
A certain level of discard of blood and its products,
particularly fresh products with a short expiry period is
both inevitable and appropriate to ensure that good quality
and safe blood products are available where and when they
are clinically necessary. However, there is a proportion of
discarding of blood and its components that is avoidable and
is called wastage. Development of strategies encompassing
various aspects of blood bank such as transport, storage and
inventory management is essential to avoid this unnecessary
wastage.2
The rate of discard of blood and its components can
be used as a quality indicator to monitor the efficiency of
the transfusion services. An increase in discard rates will
decrease the efficiency and increase the cost of production
of blood and its components. In order to decrease wastage
there is a need for better recruitment strategies, production
methods, inventory management, and recipient selection.3
Analyzing the data for discarding and the relevant reasons is
67
the first step to improve the efficiency by minimizing wastage
through measures such as training of staff. To the best of our
knowledge this is the first detailed study analysing the data
and causative factors for discarding of blood from India.
This is a retrospective study conducted over a period of 12
months from November 2012 to October 2013 in the Blood
Bank, Guru Teg Bahadur Hospital, New Delhi which is also
a Regional Blood Transfusion Center (East Delhi). A detailed
analysis of the number of blood units donated, number of
blood/blood component units discarded and the reasons for
discarding blood/blood components was carried out from
the records.
The total numbers of blood units collected during the study
period were 32980 units. Of this 24661 units were voluntary
(74.7%) and rest 8319 (25.3%) were replacement donations
(Fig. 1) from relatives of patients. The discard rate for whole
blood/RCC was 7.03% and for FFP and platelets it was 7.1 and
5.4%.
Seropositivity was the most important cause for discarding
blood and its components accounting for 54.1%, 72.7%,
55.4% of discarded RCC, platelets and FFP units respectively
(Tables 1, 3 and 5). The highest seropositivity rate was seen
for Hepatitis B which was 1.46%, followed by Hepatitis C and
HIV at 0.86 and 0.3% respectively (of all donations during the
study period).
The important causes for discarding RCC besides sero­
positivity were-expired units (17.7%), less collection (13.1%),
splitting of blood bags for pediatric patients (8.5%), donor
positive for allo or autoantibody (1.4%) and other causes
include leakage, presence of clots, hemolysis, lipemia and
over collection (Table 2).
The important causes for discarding platelets other than
seropositivity were-expired units (17.02%), RBC conta­
mination (3.5%), donor positive for allo or autoantibody (2%),
lipemic units (1.6%) and leakage of units (0.1%) (Table 4). The
important causes for discarding FFP were-leakage (18.4%),
lipemic units (8%), RBC contamination (5.7%), unused bags
246  Section 3  Blood Safety

Figure 1  Donations during study period

Table 1: Seropositivity as a cause for discarding of RCC/WB

Month Number of Number of Number of Percentage of discarded
donations discarded units reactive units reactive units
Nov 12 2402 233 100 42.9
Dec 12 2606 269 119 44.2
Jan 13 2127 281 90 32.02
Feb 13 2337 136 35 25.7
Mar 13 2476 120 92 76.6
Apr 13 2262 169 84 49.7
May 13 2321 187 107 57.2
Jun 13 2291 102 74 72.5
Jul 13 2656 160 119 74.3
Aug 13 3045 170 136 80
Sep 13 3907 247 167 67.6
Oct 13 4550 246 133 54.06
Total 32980 2320 1256
 Chapter 67  Discarding of Blood and Blood Components  247

Table 2: Reasons for discarding whole blood/RCC other than seropositivity

Month Expired Less Splitted Leakage Clotted Ab pos* Hemolyzed Other Total
collection
Nov 12 41 52 27 2 4 4 3 133
Dec 12 91 34 21 1 2 1 150
Jan 13 124 32 27 1 7 191
Feb 13 3 10 14 1 4 Lipemic -2 43
Other-9
Mar 13 4 6 13 1 1 3 0 28
Apr 13 22 23 34 1 1 2 Other-2 85
May 13 42 18 14 1 1 4 80
Jun 13 7 12 2 3 2 Lipemic-1 28
Other-1
Jul 13 1 18 13 4 1 Lipemic-1 41
Other-3
Aug 13 6 13 11 3 1 34
Sep 13 0 54 16 1 1 5 Over coll†-3 80
Oct 13 70 32 7 1 3 113
Total 411 304 199 11 12 34 6 29 1006
*- over collection, †- antibody positive in donor

Table 3: Seropositivity as a reason for discarding platelets

Month Total platelet units Number of discarded Number of reactive Percentage of discarded reactive
prepared platelet units platelet units platelet units
Nov 12 1411 79 53 67.08
Dec 12 843 64 50 78.1
Jan 13 952 125 49 39.2
Feb 13 1013 55 35 63.6
Mar 13 771 41 33 80.4
Apr 13 845 33 22 66.6
May 13 862 54 44 81.4
Jun 13* 1 1
Jul 13 1006 63 45 71.4
Aug 13 1065 53 45 84.9
Sep 13 2615 123 113 91.8
Oct 13 3945 137 113 82.4
Total 15329 828 602
* In the month of June platelets could not be made due to unfavorable weather (hot and humid) and technical reasons like faulty temperature control

after thawing (4%), donor positive for allo or autoantibody
(0.7%) and icteric bags (0.4%) (Table 6).
The blood and blood component losses occur all along the
chain of production, right from the recruitment of the donor,
bleeding of the donor, component preparation, storage of
the inventory in the blood bank, storage in the hospital or
laboratory, selection and transfusion of the recipient. Each of
these processes therefore provide an opportunity to improve
efficiency by minimising losses.3
The important causes of discarding whole blood/packed
cells included reactivity for infectious agents, expiry of blood
units, under collection and splitting of blood for pediatric
248  Section 3  Blood Safety

Table 4: Other reasons for discarding platelets

Month Expired RBC contamination Other Total
Nov 12 6 14 6 26
Dec 12 14 14
Jan 13 71 2 3 76
Feb 13 16 4 (1-Ab pos*) 20
Mar 13 5 1 2 8
Apr 13 5 2 4 11
May 13 7 3 (2-lipemic) 10
Jun 131 0 1 1
Jul 13 14 2 2 18
Aug 13 2 2 4 (4-lipemic) 8
Sep 13 2 8 (3-Ab pos*, 3-lipemic) 10
Oct 13 1 4 19 (5-lipemic, 13-Ab 24
pos*, 1-leaked)
Total 141 29 56 226
Ab pos *-donor allo or auto antibody positive

Table 5: Seropositivity as a reason for discarding FFP

Month Total FFP units Total FFP units Number of reactive Percentage of discarded reactive
prepared discarded FFP units FFP units

Nov 12 1464 67 53 79.1

Dec 12 843 95 50 52.6
Jan 13 984 76 49 64.4
Feb 13 1023 51 35 68.6
Mar 13 771 76 33 43.4
Apr 13 845 78 22 28.2
May 13 862 91 44 48.3
Jun 13 947 80 48 60
Jul 13 1080 126 45 35.7
Aug 13 1065 95 45 47.3
Sep 13 2615 166 113 68.07
Oct 13 3945 172 113 65.6
Total 16444 1173 650

patients. Other causes included leakage of blood bags,
presence of clots or antibodies, hemolysis, lipemia and over
collection.
Donor recruitment: Thorough and confidential screening,
counseling and education of blood donors will go a long way
in reducing the number of reactive blood units. Often times
this is not possible in an Indian set up because low levels of
education and awareness precludes understanding of the
reasons and justification for deferring a donation. In high
prevalence areas the use of rapid screening tests for donors
can help to reduce the wastage of donated blood.4
Blood collection: Underweight blood bags have an excess
of anticoagulant that can denature the blood during storage.
Suboptimal weight of blood collected would be unsuitable for
component preparation and transfusion. The major factors
for under collection at our centre included donor reactions
like sweating, dizziness and anxiety causing discontinuation
of the donation process, collapse of the vein and prolonged
 Chapter 67  Discarding of Blood and Blood Components  249

Table 6: Other reasons for discarding FFP

Month Leaked Unused* Ab positive Other Total
Nov 12 2 - 1 11 14
Dec 12 14 - - 31 45
Jan 13 11 6 3 7 (4-RBC † 27
3-lipemic)

Feb 13 2 6 2 6 (2-RBC †) 16
Mar 13 32 8 - 3-lipemic 43
Apr 13 25 10 - 21(9-lipemic 56
12- RBC †)

May 13 18 - 3 26 (7-RBC † 8-lipemic 47

5-icteric)
Jun 13 12 8 - 12 (5-lipemic) 32
Jul 13 16 9 - 56 (21-lipemic 81
18-RBC †)
Aug 13 17 - - 33 (27-lipemic 50
6-RBC †)
Sep 13 22 - - 31 (12-lipemic 53
15-RBC †)
Oct 13 46 - - 13 (7-lipemic 59
4- RBC †)
Total 217 47 9 250 523
*- FFP not collected from blood bank after thawing, †- RBC contamination

donation time. Most syncope attacks are vasovagal in nature
and bear no relation to the time since last meal.5 Donor
education and counseling along with a comforting and clean
environment is important to allay the anxiety surrounding
the donation process. It has been our experience that
healthy individuals hesitate to donate blood because of
the superstitions prevalent in our country regarding blood
donation, thus promoting the practice of professional donors
and middlemen. Calibrated scales and balances should be
used to accurately measure the volume of blood collected to
prevent under collection and wastage.
Small manual spring balances or scales for weighing
are still being used in mobile sessions, while blood mixing
machines are used in the blood bank. The manual spring
balances require the phlebotomist to monitor the weight
of the blood bags and mix the anticoagulant with the blood
every 60 seconds during the whole blood donation process.
Overlooking this monitoring process during busy hours can
lead to too much blood going into the collected bags. These
problems can be avoided by using the automated blood
mixing machines. When sufficient blood has entered the
bag, an audio visual alarm is activated and the blood flow
is automatically stopped by clamping the tube to prevent
further blood flow into the bag. In overweight blood bags,
the amount of anticoagulant is not enough to prevent
the blood from clotting and the clotting process may be
initiated. These blood bags with blood clots are unsuitable
for component preparation. Thus, selecting a good donor,
training and monitoring the staffs will help to reduce cases
of the underweight/overweight blood units resulting in low
discard rate.
Splitting: In the case of pediatric and thalassemic patients
requiring blood, appropriate volume bags are made by
transferring blood from the mother bag to satellite bags
through a sterile connecting device which is a closed system.6
The remaining mother bag is often discarded due to a lack of
further demand.
Leakage: The blood bag should be checked for any leakage
or defects prior to the collection of blood. Mishandling of
blood bags during collection, processing, and storage or
manufacturing error may be the major causes of defects
and leakages of blood bags. The integrity and sterility of
blood bags is mandatory and precautions should be taken
to prevent leakages. During centrifugation the bag may be
exposed to sharp surfaces or forced to a sharp wall junction
or corner, resulting in the bag material being stretched too
far, causing a tear. The defect and leakage at any part of
the plastic blood bags can be detected by visual inspection
during the processing, after pressure in a plasma extractor,
before freezing, and after thawing.
Lipemia: Gross lipemia visible on inspection was another
important cause of discarding of blood units. Some cases of
grossly lipemic components have been traced to donors who
250  Section 3  Blood Safety
had a fatty meal before donation in the study by Mbahi M
et al.7 This raises an important concern for donor screening
and education especially because donors are advised to
have adequate meals before a donation. Lipemia itself does
not affect the safety of a product but might interfere with the
ability to perform viral marker tests.8 The maintenance of a
donor registry can aid in identifying donors who previously
donated lipemic blood so that future donations can be
deferred until he/she is properly investigated.
Hemolysis: It is an important parameter for the quality
assessment of stored RBCs. It is evaluated by visual
inspection though it can be better identified by photometric
methods.  Hemolysis of red cell units occurs during processing
for component separation and also due to repeated handling
during storage, issue and transport before transfusion to
the patient.9 RBC hemolysis can be prevented by stringent
temperature regulation between 2 and 6°C, using good quality
polyvinyl chloride bags with DEHP as plasticizer which is
known to reduce hemolysis, leukocyte reduction and the use
of additive solutions containing mannitol. During component
separation, centrifugation speeds more than 5000G induce
hemolysis and this should be avoided. Transporting the
blood bags at appropriate temperature prevents hemolysis of
blood after being issued from the blood bank.10
Inventory management and expiry of blood units: Blood
bank should arrange the blood units of near expiry in front
shelves in freezers and have proper inventory management in
blood bank so that they can be used in judicious way (i.e. first in
and first out- FIFO) and wastage can be reduced. Maintaining
a record of donors of rare blood groups like Bombay blood
group who can be contacted for blood donation will help to
maintain a reserve for as and when the need arises.
To prevent wastage due to out dating a type and screen
protocol is recommended. This protocol includes screening
for other antigens apart from routine ABO and Rh typing like
ABO (A1 and A2), Rh (etc.), Bombay, Para bombay, MNS
and Kell, Lewis, Duffy and auto antibodies depending upon
protocol and incidence of other groups. If the donor has a
rare blood group which is not in demand immediately the
donation can be deferred and the donor registered with the
blood bank so that he/she can be recalled when required.
One study in Hong Kong reduced out dating rate by 11.5% to
1.3% by this protocol.11
Antibody positive: In our centre blood is screened for
antibodies post the donation process. Most of the blood units
positive for irregular antibodies are discarded because of
the lack of demand of corresponding antigen negative blood
within the expiry period of that particular unit. A system in
which donors are screened for irregular antibodies before
donation can help improve efficiency by reducing the wastage
due to presence of antibodies. Also to be emphasized in this
context is the importance of maintaining donor registries and
promoting regular voluntary donation.
Blood components like platelets and FFP have their
unique issues when it comes to wastage. One important
reason is RBC contamination which is detrimental to the
product quality. At our centre, platelets are prepared both
by PRP method and apheresis. However, for the purposes
of this study only the platelets produced by the PRP method
were considered. After centrifugation, the blood bags
should be carefully handled to prevent RBC contamination
of plasma and platelets. Subsequently, these centrifuged
blood bags should be carefully reposted onto the plasma
extractor (or blood press) and allow gentle pressure to the
centrifuged blood bag from the plasma extractor plate. The
separation should be done slowly with close monitoring
during the transfer of layers of blood components into the
satellite bags. The separation is stopped when about 8 mm of
plasma remains above the red cells.12 This technique is labor
intensive. Human factor has strong effect on the quality and
purity of the blood components preparation.
Identifying the centrifuges causing a high rate of RBC
contamination, the technician handling the centrifuge and
plasma extractor, the centrifuge speed and time, the date
and time of component separation can help to resolve the
technical details and improve the quality of the final end
product.
The short shelf life of platelets (5 days) makes it essential
to ensure a constant and proportionate production and
distribution of platelet units. This is especially tricky in
situations like dengue months where the demand outstrips
the supply and other months in the year where more platelets
are separated than the demand and hence get outdated.
FFP refers to the fluid portion of one unit of  human
blood that has been centrifuged, separated, and frozen solid
at −18 °C (−0 °F) or colder within six hours of collection. The use
of FFP has increased over the past few years due to increased
acceptance of blood component therapy. FFP can be stored
in deep freezers for prolonged duration of time (upto 1 year).
For this reason it is important to maintain an inventory and
carefully manage the limited space in a deep freeze to ensure
that a constant input output cycle is maintained so that the
near expiry units are kept in front and used first, the expired
products are discarded and space is made free for the newly
separated components. At our centre, many units of FFP
had to be discarded due to a shortage of storage space in the
deep freezer and other technical issues like power outgages.
Also other factors like decreased clinical use and demand,
non availability of plasma fractionation technique and no
permission to distribute plasma to other private centres lead
to discarding of further plasma units.
The FFP should be stored in cardboard or polystyrene
protective containers that minimize the risk of breakage
of brittle frozen product during storage, handling, and
transportation. Another approach to decrease the leakage
and contamination immediately before immersion of the
frozen blood bags in the water bath is that the whole container
should be placed in a sterile plastic bag.
 Chapter 67  Discarding of Blood and Blood Components  251
Also a few units had to be discarded because the requested
FFP unit was never collected from the Blood Bank after the
thawing procedure. Proper education of the clinicians
with regard to blood component therapy and effective
communication between the clinician and the blood bank
official is necessary to prevent such unnecessary wastage of
plasma. In addition the plasma left after the separation of
cryoprecipitate was discarded at our centre. A cryoprecipitate
is defined as the fraction of plasma obtained by slowly thawing
a single unit of FFP at 4±2 °C. This causes the cryoproteins to
precipitate out: factor VIII, fibrinogen, von Willebrand factor
(vWF), fibronect in and factor XIII. This is then centrifuged
and suspended in a small volume of plasma (30-40 mL).
Cryosupernatant or cryo-depleted plasma is the plasma
remaining after the separation of cryoprecipitate. Since there
is no demand for cryo-depleted plasma in our hospital these
units were discarded.
In a similar study conducted in Karnataka in 2009-10, the
major cause for discarding of blood units was seropositivity
for Hepatitis B (68%), followed by out dating (14.4%) and HIV
positivity (8.7%). A study conducted in Kuala Lumpur takes
into account only the quality indicators like weight of the bag,
hemolysis, leakage, etc. without considering sero positivity
which is the main cause for discarding of blood bags in our
study. In this particular study by Mohammed Morish et al.
the major reasons for discarding whole blood were under
collection and over collection, packed RBC components were
lipemia and leakage of blood bags, platelet concentrates was
red cell contamination and FFP was discarded because of
lipemia and leakages. Analysis of the discard rate of blood
and its components and the reasons thereof is an under-
recognized aspect of improving efficiency of the blood
transfusion system and the pertaining literature is scarce.
CONCLUSION
Voluntary non-remunerated donors form the bulk of blood
donors in our blood bank. Many of these voluntary donors are
motivated and are regular donors at the annually organized
camps. Considerable resources are utilized in the collection,
screening, processing and supply of safe and adequate blood
units in the health care system. Urgent measures to improve
the efficiency are warranted to address the present shortage
of blood supply in our country. There is no scope for technical
or administrative laxities when dealing with a precious
commodity like blood. Identifying and managing the reasons
for avoidable wastage of blood/components can go a long
way in improving the efficiency of blood transfusion services
without adding additional burden to the already scarce
resources in our country.
RECOMMENDATIONs
• Effective counseling and self-deferral of donors with high-
risk behavior can reduce the collection of blood from
seroreactive donors.
• An effective watchful stock management system can
reduce the wastage of red cell due to expiry.
• The BTS should be fully computerized with software on
blood management system which can notify the expiry of
a blood unit beforehand.
• The government should plan to setup a plasma fractiona­
tion center so that excess plasma can be sent for compo­
nent separation.
• Effective training of resident doctors and technical staff
will go a long way in preventing unnecessary wastage of
blood bags.
• Regular appointment of phlebotomist in place of resident
doctors who comes only for a period of one to six months.
REFERENCES
1. Kora SA, Keshav K. An analysis of donor blood wastage in
a blood bank in rural Karnataka. Journal of Clinical and
Diagnostic Research. 2011;Suppl-2,5(7):1393-6.
2. National blood and blood product wastage reduction strategy
2013-2017. National Blood Authority, Australia.
3. Cobain TJ. Fresh blood product manufacture, issue, and use: a
chain of diminishing returns? Transfus Med Rev.  2004;18(4):
279-92.
4. Lubna N, Haque AU. Predonation testing of the potential blood
donors—A pilot study at a tertiary care hospital.  Int J Pathol.
2009;7(1):25-30.
5. Naveen A,  Neelam M,  Ratti RS. Analysis of adverse events
and predisposing factors in voluntary and replacement whole
blood donors: A study from North India. Asian J Transfus
Sci. 2012;6(2):155-60.
6. Burghardt DC. Component preparation and storage. In:
Hillyer C, Strauss R, Luban N, editors. Handbook of Pediatric
Transfusion Medicine. Academic Press; 2004. pp. 15-6. 
7. Mbahi M, Reddy V, Narvios A. Transfusion medicine history
illustrated. Milky platelet concentrate: a second look Trans­
fusion. 2006;46(6):877.
8. Visual Inspection Reference Guide.  American National Red
Cross. 2006. pp. 6-26.
9. Sawant RB, Jathar SK, Rajadhyaksha SB, Kadam PT. Red cell
hemolysis during processing and storage. Asian J Transfus Sci.
2007;1:47-51.
10. Choudhury N, Mathur A. Visual detection of hemolysis in
blood bag before issue. Asian J Transfus Sci. 2011;5(1):61-2.
11. Feng CS, Ng AK. An analysis of donor blood wastage due to
outdating in a large teaching hospital. Pathology 1991;23(3):
195-7.
12. Mohammed M,  Yasmin A. Quality indicators for discarding
blood in the national blood center, Kuala Lumpur. Asian J
Transfus Sci. 2012;6(1):19-23.
Adverse Transfusion Reactions

Manoj A Kahar
68
INTRODUCTION Diseases simulating HTR include Paroxysmal Nocturnal
Hemoglobinuria, Autoimmune hemolytic anemia, G-6-P-D
Blood transfusion like administration of any other drug is an deficiency, Malignant Hyperthermia, Hemoglobinopathies
irreversible event that carries potential benefits and risks to and RBC membrane defects.
the recipient. Some typical causes of transfusion associated deaths are:
Definition of adverse transfusion reaction: Any unfavorable • Acute hemolysis (ABO-incompatible blood components)
transfusion – related event occuring in a patient during or • Acute pulmonary edema
after transfusion of blood components. • Bacterial contamination of product
• Delayed hemolytic reactions
• External hemolysis (e.g. temperature exceeded 40°C)
Classification
• Acute hemolysis; damaged blood component (e.g.
Adverse transfusion reaction (ATR) may be immediate nondeglycerolized, improper solution)
(occuring during or within 24 hrs after completion of • Transfusion-associated graft-versus-host disease.
transfusion) or delayed (evident after days, weeks, months The three leading causes of transfusion-related death
or even years after transfusion). Immediate and delayed reported in literature are transfusion related acute lung injury
transfusion reactions are further divided as immune (TRALI), HTRs and bacterial contamination.
(mediated by Antigen-Antibody reaction) or nonimmune.
Pathophysiology of immune hemolysis: In vivo lysis of
Reaction Immediate (Acute) Delayed RBCs can result from the interaction of complements with the
Immune IHTR DHTR RBC membrane (intravascular hemolysis) (Flow chart 1) or
mediated FNHTR PTP the removal of cells sensitized with IgG and/or complement
Allergic TA-GVHD by the reticuloendothelial system (extravascular hemolysis).
Anaphylactic and Anaphylactoid
NCPE-TRALI ACUTE Adverse transfusion reaction
Non immune Bacterial contamination Iron overload WITH IMMUNE CAUSES
mediated Circulatory overload Transfusion
Physical or chemical damage to associated Immediate Hemolytic Transfusion Reaction
RBCs infections
Depletion and dilution of Immediate hemolytic transfusion reaction (IHTR) occurs
coagulation factors and platelets very soon after the transfusion of incompatible RBC. The RBC
is rapidly destroyed, releasing hemoglobin and RBC stromata
It may be impossible to assess the severity of ATR by the in the circulation (Flow chart 2).
symptoms because a life-threatening (acute hemolytic) and • Cause: The majority of hemolytic reactions are caused by
relatively mild (nonhemolytic febrile) transfusion reaction transfusion of ABO incompatible blood, e.g. group A, B or
may initially cause exactly the same symptoms (e.g. fever, AB red cells to a group O patient. Most hemolytic reactions
chills). Hence, any ATR should be considered a potentially are the result of human error such as the transfusion of
life-threatening reaction until clinical observations and/ properly labeled blood to the wrong patient, or improper
or laboratory results establish otherwise. On the other side, identification of pre-transfusion blood samples.
there are many diseases that can cause RBC hemolysis and • Non-immune hemolysis of RBCs in the blood container or
misinterpreted as hemolytic transfusion reaction (HTR). during administration can occur due to physical disruption
 Chapter 68  Adverse Transfusion Reactions  253

(temperature changes, mechanical forces, non-isotonic Flow chart 1  Complement activation may be initiated by antibody
fluid). binding to the appropriate antigens on the red cells
• Symptoms: Chills, fever, pain (along IV line, back, chest),
hypotension, dark urine, uncontrolled bleeding due to
DIC.
• Management:  Immediately stop transfusion. Notify
hospital blood bank urgently (another patient may also
have been given the wrong blood!). These patients usually
require ICU support and therapy includes vigorous treat-
ment of hypotension and maintenance of renal blood flow.
• To assess the patient’s condition, several laboratory
parameters should be monitored during the next days:
Electrolytes, ABG, Coagulation tests, CBC and Creatinine.
• Prevention:  Proper identification of the patient from
sample collection through to blood administration, proper
labeling of samples and products is essential (Figs 1 to 3).
Prevention of non-immune hemolysis requires adherence
to proper handling, storage and administration of blood
products.

Flow chart 2  Mechanism of intravascular RBC destruction

254  Section 3  Blood Safety
Figure 1  Commercially manufactured identification systems.
Compare preprinted label with the patient armband
Figure 2  Confirm that sample and requisition form agree
Figure 3  Commercially manufactured identification systems. Pre-
transfusion comparison of recipient armband and donor unit bag
INVESTIGATIONS TO BE DONE FOR
immediate hemolytic transfusion
reaction
• Take blood for
– CBC, plasma hemoglobin
– Direct anti-globulin test, ABO and Rh group
– Repeat Cross-match
– Coagulation screen—PT, PTT, fibrinogen
– Chemistry—creatinine, electrolytes
– Blood cultures
• Return blood pack(s) and giving set to the Blood Bank.
• Insert bladder catheter and monitor urine output. Give
frusemide if no diuresis.
• Additional blood tests required to monitor the course of
the IHTR.
• If bacterial contamination is suspected treat with broad-
spectrum intravenous antibiotics.
• If disseminated intravascular coagulation (DIC) develops
give blood components guided by clinical state and
coagulation screen results.
• If the patient needs further transfusion use matched blood.
Febrile Nonhemolytic Transfusion Reactions
Definition: AABB Technical Manual defines FNHTR as
a 1°C temperature rise associated with transfusion and
having no medical explanation other than blood component
transfusion.
Other Definitions include:
• Any 1°C or greater temperature increase above the
patient’s baseline temperature, during or within 24 hrs
after transfusion, with a minimum recorded temperature
of 38°C.
• A 1°C temperature increase above the patient’s baseline
temperature, during or within 8 hrs after the end of the
transfusion.
– Cause: Fever and chills during transfusion are thought
to be caused by recipient antibodies reacting with
white cell antigens or white cell fragments in the blood
product or due to cytokines which accumulate in the
blood product during storage. Fever occurs more
commonly with platelet transfusion (10–30%) than red
cell transfusion (1–2%).
– It is important to distinguish from fever due to the
patient›s underlying disease or infection (check
pretransfusion temperature). Fever may be the initial
symptom in a more serious reaction such as bacterial
contamination or hemolytic reaction.
– Multiparous women and those who have received
multiple transfusions are most at risk.
– Usually symptoms develop towards the end of
transfusion or in the subsequent 2 hours.
– Management: Symptomatic, paracetamol.

– Investigation:  Fever can be the initial sign in more
severe transfusion reactions (hemolytic or bacterial
sepsis) and should be taken seriously.
– For isolated fever or chills in some patients, the medical
officer may elect to restart the transfusion. If the fever is
accompanied by significant changes in blood pressure
or other signs and symptoms, the transfusion should be
ceased and investigated.
– Check for HLA antibodies in patients having repeated
febrile reactions.
– Prevention: A proportion of patients who have febrile
reactions will have similar reactions to subsequent
transfusions. Many are prevented by leucocyte filtration
(either bedside or pre-storage).
Urticarial (Allergic) Reactions
• Cause: Seen in approximately 1% of recipients and caused
by foreign plasma proteins. On rare occasions they may
be associated with laryngeal edema and bronchospasm.
Two possible etiologies have been proposed, based on the
passive transfer of donor plasma (1) The donor plasma has
an allergen with which IgE, IgG or both antibodies in patient
plasma react. (2) The donor plasma has regains (IgE or IgG
or both) that combine with allergens in the patient plasma.
• Management:  If urticaria occurs in isolation (without
fever and other signs), slow the rate or temporarily stop
transfusion. If symptoms are bothersome, consider
administering an antihistamine before restarting the
transfusion. If associated with other symptoms, cease the
transfusion and proceed with investigation.
• Investigation:  In the case of mild urticarial reactions
with no other signs or symptoms, it is not necessary to
submit blood specimens for investigation. It is also usually
possible to restart the transfusion. Such a decision should
be made after assessment by the treating doctor.
Anaphylactic and Anaphylactoid Reactions
• Anaphylactic and anaphylactoid reactions have signs
of cardiovascular instability including hypotension,
tachycardia and loss of consciousness, cardiac arrhythmia,
shock and cardiac arrest. Sometimes respiratory
involvement with dyspnea and stridor are prominent.
• Cause:  In some cases patients with IgA deficiency that
have anti-IgA antibodies can have these reactions.
• Management:  Immediately stop transfusion, supportive
care including airway management may be required.
Adrenaline may be indicated. Usually given as 1:1000
solution, 0.01mg/kg s.c./i.m. or slow i.v.
• Investigation: IgA levels and anti-IgA antibodies.
• Prevention:  Patients with anti-IgA antibodies require
special blood products such as washed red blood cells
and plasma products prepared from IgA deficient donors.
Manage further transfusion in consultation with the
hematologist-on-call.
Chapter 68  Adverse Transfusion Reactions  255
Transfusion Related Acute Lung Injury
Synonyms: Non-cardiogenic pulmonary edema (NCPE),
Pulmonary hypersensitivity reaction and allergic pulmonary
edema.
• Transfusion related acute lung injury (TRALI) is a clinical
diagnosis of exclusion characterized by acute respiratory
distress and bilaterally symmetrical pulmonary edema
with hypoxemia (PaO2/FiO2 < 300 mm Hg), normal central
venous pressure and normal pulmonary wedge pres­
sure developing within 2 to 8 hours after a transfusion.
A CXR shows interstitial or alveolar infiltrates when no
cardiogenic or other cause of pulmonary edema exists
(Figs 4A and B).
• Cause:  Pulmonary vascular effects are thought to occur
secondary to cytokines in the transfused product or from
interaction between patient white cell antigens and donor
antibodies (or vice versa).
• Predisposing factors for TRALI include: Direct lung injury
(aspiration, pneumonia, toxic inhalation, lung contusion,
near drowning) and Indirect lung injury (sepsis, shock,
multiple trauma, burn injury, acute pancretitis, CABG,
drug overdose)
• Management:  Symptomatic support for respiratory
distress includes oxygen administration and 72% cases
may require intubation and mechanical ventilation.
Symptoms generally resolve over 24–48 hours. Death
occurs in 5–10% of patients experiencing a TRALI reaction.
• Suggested prerequisites for performing a laboratory
workup:
– A laboratory workup of a TRALI case is expensive.
– Donors may experience anxiety and/or concern that
their blood donation may have resulted in harm to a
recipient. Antibodies are most common in multiparous
female donors as consequence of prior pregnancies.
A B
Figures 4A and B  Chest X-ray of a patient before and during an
episode of transfusion-related acute lung injury
256  Section 3  Blood Safety
– For these reasons, the transfusing institution has the
obligation of providing complete information on the
TRALI case to the blood center.
• Information to be provided should include:
– Sufficient clinical data to confirm the TRALI diagnosis
and to exclude other causes of ALI.
– The results of any laboratory tests performed to rule out
other transfusion reactions.
– Data as to the storage age of the transfused components
for evaluating the neutrophil priming hypothesis of
TRALI pathogenesis.
ACUTE Adverse transfusion reaction
WITH NONIMMUNE CAUSES
Transfusion Associated Circulatory Overload
• Transfusion associated circulatory overload (TACO) is
a good example of an iatrogenic transfusion reaction.
Circulatory overload results from impaired cardiac
function and/or excessively rapid rate of transfusion.
• Patients at significant risk include patients over 60 yrs of
age, infants, patient with severe euvolemic anemia (Hb
< 5 gm/dL), cardiac disease, thalassemia and sickle cell
disease.
• Incidence: Current estimate of the frequency of TACO is
1 in 700 recipients; in perioperative surgery setting in older
orpthopedic patients, incidence is much higher 1 in 100
patients.
• Clinical presentation: Dyspnea, orthopnea, cyanosis,
tachycardia, increased venous pressure and hypertension.
• Cause: Patients with cardiopulmonary disease and infants
are at risk of volume overload especially during rapid
transfusion.
• Management:  Stop the transfusion; administer oxygen
and diuretics as required.
• Prevention:  Pre-transfusion assessment is important
to identify patients at risk and management should be
adjusted accordingly. Preventive measures include, trans­
fusion over longer periods (maximum 4 hrs), pre-emptive
diuretics and components can be split into smaller
aliquots to further reduce the speed of infusion without
washing product or increasing donor exposure, in patients
at risk avoid transfusing more than one unit at a time.
Bacterial Contamination (Infective Shock)
• Cause: Bacteria may be introduced into the pack at the time
of blood collection from sources such as donor skin, donor
bacteremia or equipment used during blood collection or
processing. Bacteria may multiply during storage. Bacteria
commonly implicated are those capable of growing in
cold temperatures (Psychrophilic) such as Pseudomonas
species, Escherichia coli and Y. enterocolitica. Bacterial
sepsis accounts for atleast 10% of transfusion-associated
fatalities. Platelets are more frequently implicated than
red cells because of their storage at 20-24°C.
• Symptoms: Very high fever, rigors, profound hypotension,
nausea and/or diarrhea.
• Management:  Immediately stop the transfusion and
notify the hospital blood bank. After initial supportive
care, blood cultures should be taken and broad-spectrum
antimicrobials commenced. Laboratory investigation will
include culture of the blood pack.
• Prevention:  Inspect blood products prior to transfusion.
Some but not all bacterially contaminated products can be
recognized (clots, clumps, or abnormal color). Maintaining
appropriate cold storage of red cells in a monitored blood
bank refrigerator is important. Transfusions should
not proceed beyond the recommended infusion time
(4 hours).
Physically or Chemically Induced
Transfusion Reactions
Definition: Physically of chemically induced transfusion
reactions (PCITRs) are a heterogeneous group and includes
physical RBC damage, depletion and dilution of coagulation
factors and platelets, hypothermia, citrate toxicity,
hypokalemia or hyperkalemia and air embolism. Because the
clinical signs and symptoms of PCITRs can be suitable, the
transfussionist must be alert to identify the reaction effects.
Hypothermia
Cause: Rapid infusion of large volumes of stored blood (cold
fluid transfusions) contributes to hypothermia (core body
temperature lower than 35°C. Infants are particularly at risk
during exchange or massive transfusion.
Hypothermia inhibits the immune system function,
intensifies lactic acidosis and cardiac arrhythmias and can
cause coagulopathies.
Prevention and management:  Appropriately maintained
blood warmers should be used during massive or exchange
transfusion. Additional measures include warming of other
intravenous fluids and the use of devices to maintain patient
body temperature.
Citrate Toxicity
• Cause: Citrate is the anticoagulant used in blood
products. It is usually rapidly metabolized by the liver.
Rapid administration of large quantities of stored blood
may cause hypocalcemia and hypomagnesemia when
citrate binds calcium and magnesium. This can result
in myocardial depression or coagulopathy. Patients
most at risk are those with liver dysfunction or neonates
with immature liver function having rapid large volume
transfusion.

• Management: Slowing or temporarily stopping the trans­
fusion allows citrate to be metabolised. Replacement
therapy may be required for symptomatic hypocalcemia
or hypomagnesemia.
Potassium Effects
• Cause:  Stored red cells leak potassium proportionately
throughout their storage life, transfusion of such red cells
can lead to hyperkalemia. After 28 days storage in citrate, a
unit of RBC contains approximately 6 mmol of potassium
per unit. Irradiation of red cells increases the rate of
potassium leakage. Clinically significant hyperkalemia
can occur during rapid, large volume transfusion of older
red cell units in small infants and children. Transfusion
induced hypokalemia is most likely to be caused infusion of
intracellular potassium depleted RBC blood components,
such as usual RBCs or frozen washed RBCs.
• Prevention: Blood less than 7 days old is generally used
for rapid large volume transfusion in small infants (e.g.
cardiac surgery, exchange transfusion).
DELAYED Adverse transfusion
reaction WITH IMMUNE CAUSES
Delayed Hemolytic Transfusion Reaction
• Definition: Delayed hemolytic transfusion reaction (DHTR)
is most often the result of an anamnestic response in a
patient who has previously been sensitized by transfusion,
pregnancy, or transplant and in whom antibody is not
detectable by standard pretransfusion methods.
• Cause: A DHTR occurs when a patient develops an
antibody directed against an antigen on transfused
red cells. Antibodies implicated in DHTR are Anti JKa,
Anti-E, Anti-D, Anti-C, Anti-K, Anti Fya, Anti M (common
antibodies) and Anti-A1, Anti-P1 (uncommon antibodies).
The antibody may cause shortened red cell survival,
with clinical features of fever, jaundice and lower than
expected hemoglobin following transfusion. Most delayed
hemolytic reactions produce few symptoms and may
go unrecognized however there are reports of serious
consequences in critically ill patients.
• Prevention: An antibody screen is performed as part of
pre-transfusion testing. When an antibody is detected,
it is identified and appropriate antigen negative blood is
provided. Sometimes antibodies fall below detectable
limits and may not be detected by pretransfusion testing.
A medical alert card should be issued to all patients in
whom irregular antibodies were found so that they can
present this information at the time of transfusion in a
different medical facility.
Alloimmunization
• Patients experiencing alloantibody formation are
asymptomatic. The alloantibody is discovered at the time
Chapter 68  Adverse Transfusion Reactions  257
of pretransfusion testing. Appropriate antigen negative
blood will be supplied.
• Prevention: Alloimmunization to the D and K (Kell)
antigens is prevented by the provision of Rh(D) negative
and Kell negative blood for Rh(D) negative, Kell negative
patients. This is important for females with child-bearing
potential as these antibodies can cause severe hemolytic
disease of the newborn during pregnancy.
• At risk groups: Patients with sickle cell disease or major
hemoglobinopathy syndromes who are chronically
transfused are at greatest risk of alloantibody formation.
Prior to commencing transfusion, patients with these
condition should have extended red cell phenotyping
performed (EDTA sample). Blood matched for the
patient’s Rhesus and Kell antigens is usually supplied for
transfusion.
Platelet Refractoriness
• When thrombocytopenic patients do not achieve the
expected post-transfusion platelet count increment they
are said to be refractory. This usually occurs in patients
receiving frequent platelet transfusions.
• Cause:
– Clinical causes include; sepsis, DIC, bleeding, fever,
some drugs, and enlarged spleen.
– Immunological causes include; the development of
antibodies to human leucocyte antigens (HLA) or
human platelet antigens (HPA).
• Management: Immunological refractoriness can be mana­
ged by the provision of HLA or HPA matched platelets.
• Prevention:  Leukocyte reduction of blood products
to levels less than 106/unit reduces the likelihood of
alloimmunization. This can be achieved through the use of
prestorage or bedside leucocyte reduced blood products.
Post Transfusion Purpura
• It is characterized by a rapid onset of thrombocytopenia
due to anamnestic production of platelet alloantibody.
• Post transfusion purpura (PTP) usually occurs in multi­
parous females. The lag time between transfusion and
onset of thrombocytopenia is approximately 7 to 14 days.
• Purpura and thrombocytopenia occur about 1 to 2
weeks after transfusion. Hematuria, melena, and vaginal
bleeding have also been reported.
Pathophysiology:
– Platelet antibody specificity most frequently identified
is HPA-1a (anti-PLA1), others are HLA-A2 and
Lymphocytotoxic antibodies.
– Platelet alloantibody attaches to the platelet surface
which permits extravascular destruction by the RES in
the liver and spleen. The patient’s autologous platelets
are destroyed enhancing the thrombocytopenia.
Management: Three types of therapy have been advocated:
Corticosteroids, exchange transfusions, and plasmapheresis.
258  Section 3  Blood Safety
Intra­
venous immunoglobulin therapy has also been
advocated.
Transfusion-Associated Graft Versus
Host Disease
• Cause:  Transfusion-associated graft versus host disease
(TA-GVHD) occurs when donor lymphocytes in cellular
blood products engraft in a susceptible transfusion
recipient. These donor lymphocytes proliferate and
damage target organs especially bone marrow, skin,
liver and gastrointestinal tract. The clinical syndrome
comprises fever, skin rash, pancytopenia, abnormal liver
function and diarrhea and is fatal in over 80% of cases with
a median survival of 21 days after transfusion. The usual
onset is 8–10 days post transfusion, with a longer interval
between transfusion and onset of symptoms in infants.
• The most commonly reported setting for Ta-GVHD is
immunocompetent recipients of blood from biologically
related (directed) or HLA identical donors. The disease
is also reported in immunologically compromised
patients. Death is usually due to infection or hemorrhage
secondarily to bone marrow aplasia.
• Pathophysiology: GVHD is caused by a proliferation
of T-cell lymphocytes derived from the donor blood
immunologically responding to major and minor histo­
com­patibility antigens in the patients. Patients with cell
mediated immunodeficiency are at risk of not being able
to reject transfused lymphocytes. Another risk group is
patients haploidentical with that of the donor, these are
usually first degree of reactions. Diagnosis can be made
by skin biopsy, liver biopsy or bone marrow examination.
HLA typing essential to confirm the diagnosis.
• Prevention: Gamma irradiation of cellular blood products
(whole blood, red blood cells, platelets, granulocytes) for
at risk patients.
Immunomodulatory Effects
• Some studies suggest a link between blood transfusion
and increased risk of infection and cancer recurrence.
However, this is currently considered unproven.
• Cause: Unknown, possibly mediated by donor white cells
or plasma.
• Management and prevention:  Not known, possibly
leucocyte depletion of blood products.
DELAYED Adverse transfusion
reaction WITHOUT IMMUNE CAUSES
Iron Accumulation
Cause:  Iron accumulation is a predictable consequence
of chronic RBC transfusion. Organ toxicity begins when
reticuloendothelial sites of iron storage become saturated.
Liver and endocrine dysfunction creates significant morbidity
and the most serious complication is cardio toxicity which
causes arrhythmias, and congestive heart failure. Patients
receiving chronic transfusion usually have their iron status
monitored and managed by their physician.
Management and prevention:  Iron chelation therapy is
usually commenced early in the course of chronic transfusion
therapy.
Infectious Disease Transmission
• A variety of infectious agents may be transmitted by
transfusion. Definitive evidence of transmission by
transfusion requires demonstration of seroconversion or
new infection in the recipient and isolation of an agent
with genomic identity from both the recipient and the
implicated donor.
• Strong presumptive evidence of transfusion transmission
includes recipient seroconversion within an appropriate
interval after transfusion, the recognition of appropriate
infectious markers in an implicated donor on follow-up
investigation, or both.
• Suspected transfusion-transmitted bacterial or parasitic
infection (malaria) should be reported urgently in order to
recall other potentially infectious blood products from the
same donation.
• The risk of acquiring an infectious disease through blood
transfusion has not been totally eliminated even though
the level and sensitivity of testing today makes transfusion
very safe. Physicians/hospital staff should report all
instances when an infectious disease is reasonably likely
to have been transmitted by a blood transfusion. 
• The requirements for investigating transfusion associated
infections (TAI) are found in the Code of Federal
Regulations, Title 21, Section 606.170, and the AABB
Standards.
• The most commonly investigated infectious diseases are:
retroviruses, hepatitis viruses, West Nile virus, malaria,
babesiosis, and Chagas disease.
The transfusion center investigation includes:
• Complete medical history
• Clinical laboratory data
• History of known risk factors for the reported infection.
The objectives of blood center investigations of Tai are:
• Quarantine of additional infectious components
• Deferral of an “unsafe” donor
• Notification of the donor and other recipients of the
donor’s blood
• Process improvements
• Given the risks related to transfusion practice, it is of
paramount importance to have adequate skills and to take
optimal precautions for the transfusion of blood and blood
components.

CONCLUSION
The following questions must be raised
• Is there a need for the transfusion?
• What are the real needs of the patient? Which blood
component should be chosen?
• How much is needed and how often?
• How can the transfusion be administered optimally?
A transfusion should never be ordered or given, unless it is
worth the risk.
Karl Landsteiner
REFERENCES
1. Technical Manual, 15th edn, Bethesda, Maryland, American
Association of Blood Banks, 2005.
2. Denise M. Harmening, Modern Blood Banking and Transfusion
Practices, 5th edn, Philadelphia, F.A. Davis Company, 2008.
Chapter 68  Adverse Transfusion Reactions  259
3. Eva D. Quinley, Immunohematology Principles & Practice, 3rd
edn, Lippincott Williams & Wilkins, 2011.
4. Daniels G, Bromilow I. Essential Guide to Blood Groups, 1st
edn, Blackwell Publishing Ltd., USA 2007.
5. Sally V. Rudmann, Textbook of Blood Banking and Transfusion
Medicine, 2nd edn 2005, Elsevier Saunders.
6. Petz LD, Garatty G. Immune Hemolytic Anemias, Philadelphia,
Churchill Livingstone, 2004.
7. Poole J, Daniels G. Blood groups antibodies and their
significance in transfusion medicine, Transfusion. Med Rev.
2007;21(1):58-71.
8. Callum J, Pinkerton P. A guide to Transfusion Medicine, 2nd
edn, 2006.
9. Strobel E. Hemolytic Transfusion Reactions, Transfusion Med.
Hemother. 2008;35(5).
10. Adverse Transfusion Reactions; As defined in CDC’s National
Healthcare Safety Network Hemovigilance Module (www.
aabb.org/biovigilance & www.cdc.gov/nhsn/bio.html).
Hemovigilance and its
Launch in India
Neelam Marwaha
Introduction
Blood and its components prescribed by almost all medical
and surgical specialists, are considered life-saving but may
give rise to undesirable and unintended adverse effects.
Hence, the safety of the entire transfusion chain, i.e. from
the donor to the recipient needs monitoring. This can only
be achieved by careful observation and analysis of adverse
events, hence hemovigilance, which is defined as “a set of
surveillance procedures, from the collection of blood and
its components to the follow-up of recipients to collect and
assess information on unexpected or undesirable effects
resulting from the therapeutic use of labile blood products
and to prevent their occurrence or recurrence”.
The objectives of hemovigilance are:
• To recognize serious adverse events of transfusion
• To analyze factors leading to such events.
• To devise preventive strategies.
• To analyze the outcome of preventive strategies.
Hemovigilance systems
Hemovigilance in a country can be organized in different
ways. However, for logistic reasons, the system has been
organized based on the existing network for collection of data
related to blood transfusion services in various countries.
There are at least three distinct patterns of hemovigilance.
1. Through legislation.
2. Voluntary reporting of transfusion events.
3. Multidisciplinary regulation.
Outcome of Hemovigilance
around the world
As a result of increasing implementation of hemovigilance
in various countries the following observations have been
made:
69
• Bacterial contamination of blood and blood products
constituted a more frequent infectious hazard of blood
transfusion rather than viral diseases.
• The risk of transmitting viral infection by use of screened
blood is almost negligible and is mainly linked to donations
occurring in the window period, when sensitive testing
technologies are applied.
• Administration of ABO incompatible blood was invariably
due to failure to detect incorrect identity of patient or the
blood unit. Errors occurred both in the blood centre and
the patient bedside, the latter being more common, i.e.
failure to check patient and blood unit identity at bedside.
• Administration of ABO compatible but non-compatibility
due to other alloantibodies occurred on two accounts (a)
failure of clinicians to provide information regarding prior
transfusions and (b) technical limitations of alloantibody
detection and typing systems.
• Immune non-hemolytic complications of transfusion
like transfusion related acute lung injury (TRALI), post
transfusion purpura (PTP) and transfusion-associated
graft versus host disease (TA-GVHD) were unpredictable
and seemed under reported.
• The causes of transfusion related mortality were TA-GVHD,
TRALI, bacterial contamination of blood components
and incorrect blood/component transfused. Bacterial
contamination emerged as a cause for concern.
• Pre-deposit autologous transfusion also could not
guarantee 100% safety as there was occurrence of clerical
errors leading to ABO mismatched blood issue and
bacterial contamination on storage.
Recommendations and Initiatives
Based on analysis of hemovigilance data various countries
where such a system has been established have developed
transfusion guidelines to minimize serious hazards of
transfusion.

• Transfusion errors: “Getting the right blood/component
to the right patient at the right place at the right time” is
a complex organizational process that involves patient’s
bedside sampling through laboratory testing, issuing of
blood/component and finally bedside transfusion. Errors
may occur at any stage of the process. Each hospital/
institution needs to formulate/adopt guidelines where
patient identification and blood/component identification
are made in accordance with strict procedures. The
guidelines need to be circulated to all user departments/
clinicians periodically. Use of the computerization in
transfusion services with barcode printers and scanners
to provide bar-coded identities for patient’s samples
and blood packs and introduction of an IT “block” to the
administration of blood without confirming identity of
patient and blood unit were suggested. Implementation
of computerization has definitely reduced the risks of
transfusion errors.
• Bacterial contamination: Careful donor screening and
proper phlebotomy site preparation are the first essential
prerequisites. Storage at recommended temperatures and
specified shelf life must be observed. Visual inspection
of blood/component unit before issue and also before
transfusion is necessary. Bacterial screening of platelet
concentration is also being implemented. Automated
rapid bacterial detection systems have been developed.
• Immune complications: Clinician’s awareness regarding
TRALI and TA-GVHD needs to be increased. Appropriate
diagnostic guidelines need to be formulated particularly
for TRALI. Hemolytic transfusion reactions other than
those due to ABO system antigens can best be avoided by a
type and antibody screen rather than type and cross match
policy.
• New blood safety initiatives: Pathogen inactivation
of fresh frozen plasma and platelet concentrates will
take care of window phase transmissions and emerging
infectious threats. Clinical trials are being conducted to
evaluate product safety and efficacy.
“Near Miss” events are errors which are detected before
the transfusion of incorrect blood/component, and hence a
major transfusion error can be avoided. Reporting of “Near
Miss” events helps in identifying “hot spots” for transfusion
errors and is of immense value for hemovigilance purposes.
The objective of a Rapid Alert System is to take corrective
measures in the shortest possible time. It has been used to
alert the appropriate authorities to take note of appearance
of clusters of transfusion reactions, defects in disposable
materials in the transfusion chain or defective equipments,
etc. To cite an example in most of the tertiary care hospitals in
our country, blood components are transfused in preference
to whole blood. The shelf-life of each component is different;
hence the time of actual transfusion of each component from
the same donor may be different. Platelets have a short shelf-
life (3 to 5 days) while red cells have shelf-life of 35 days and
Chapter 69  Hemovigilance and its Launch in India  261
fresh frozen plasma and cryoprecipitate for one year. In case
platelets have been detected to have bacterial contamination,
a rapid alert to the blood center will lead to immediate
removal of other components from that donor unit from the
inventory.
The plasma derivatives like albumin, factor concentrates
and immunoglobulin preparations which are prepared
through plasma fractionation are covered under pharma­
covigilance, in most of the countries.
Donor reactions have a negative impact on voluntary
blood donation programme. Such information is relevant for
formulating appropriate donor motivational strategies and
post-donation care of donors. Donor vigilance is another
important area and definitions of donor reactions have
already developed by the ISBT working party.
India had no national hemovigilance system in place till
the year 2012, although the implementation of hemovigilance
program is included in the National Blood Policy. Few
institutional efforts at hemovigilance have been reported in
the scientific literature from the country, but that does not
constitute a system.
Hemovigilance in India
A centralized and structured hemovigilance programme was
launched in India on 10th December 2012. It is an independent
programme under the broad ambit of Pharmacovigilance
Programme of India (PvPI), which was initiated in July 2010
with the objective of assuring patient safety and promoting
public health. The PvPI is being executed by the Indian
Pharmacopoeia Commission, Ministry of Health and Family
Welfare (MOHFW), Govt. of India. The data is being collected
through adverse drug reaction (ADR) monitoring centers set
up in 90 medical institutions in the country. Trained staff- the
technical associates, have been recruited for these ADRs for
data collection and submission. After the successful launch
of PvPI, the hemovigilance programme has been started
as a collaborative venture by the Indian Pharmacopoeia
Commission and the National Institute of Biologicals (NIB),
an autonomous institute under the MOHFW, Govt. of India,
which ensures quality of biologicals for use in the country. The
National Co-ordinating Center for hemovigilance is located
at NIB, and a Core Committee chaired by the Director, NIB,
co-ordinates this programme.
The objectives of the programme are (i) to collect, collate
and analyse data related to transfusion reactions, (ii) to create
awareness amongst healthcare professionals in the country
for participation in the programme, (iii) to generate evidence
based recommendations and forward the same to central
drugs standards control organisation (CDSCO) for blood
safety related regulatory decisions, (iv) to communicate
relevant information to all key stakeholders and (v) to create
national and international linkages.
The Core Committee is assisted by a National Advisory
Committee which has a varied representation, reflecting
262  Section 3  Blood Safety
key stakeholders. After detailed discussions amongst all the
committee members, the transfusion reaction reporting
form (TRRF) and a guidance document for filling the
TRRF have been finalized. Reporting has been initiated for
severe transfusion reactions as per ISBT reportable table of
serious adverse events so as to harmonize definitions as per
international norms. Each reporting forms also documents
the imputability assessment of the reaction. Software has
been developed by the IT division of NIB and validated for
online submission of TRRF from medical institutions. The
responsibility for submission of hemovigilance data has
been given to the medical officer in charge of blood centers
/departments of transfusion medicine with assistance from
the technical associates from the PvPI.
Presently, reporting is voluntary and limited to information
related to adverse transfusion reactions. Awareness about the
programme, its objectives and its non-punitive implications
is being generated through a hemovigilance newsletter
accessible at website hemovigilance@nib.gov.in and also
through a series of workshops to be held in different regions
of the country.
The functional units for hemovigilance in a particular
center as defined in the guidance document are as follows.
• Medical and nursing staff at the patient bedside
• Department of transfusion medicine
• Hospital transfusion committee
• Technical associate IPC-PvPI
• Hemovigilance center, NIB
• PvPI national co-ordinating center, IPC
• CDSCO
The entire programme has been structured to collect,
collate and analyse data for making recommendations to the
regulators to make policy changes for improving blood safety.
Roadmap for Hemovigilance in the Country
The programme is planned to proceed in a phase-wise
manner. An initiation phase (2012-13), an expansion and
consolidation phase (2013-15), an expansion and mainte­
nance phase (2015-16) and optimization phase (2017).
An Initiation Phase (2012-13)
• Develop systems and procedures for data collection
• Enroll 90 medical colleges
• Initiate software development for national hemovigilance
database
• Start data collection
• Development and establishment of training centers
• Training of hemovigilance human resource
• Zonal workshop for public awareness of transfusion safety
• Initiate and continue publication of hemovigilance
newsletter.
An Expansion and Consolidation
Phase (2013-15)
• Enroll additional 10 medical colleges
• Continue training of hemovigilance human resource
• Identify gaps and address through appropriate training
• Link up with international hemovigilance network.
An Expansion and Maintenance
Phase (2015-16)
• Enroll all remaining medical colleges
• Continue training of hemovigilance human resource.
Optimization Phase (2017)
• Create center of excellence for hemovigilance.
Till date the progress in this programme has been highly
promising. The transfusion reaction reporting form (TRRF),
a Guidance Document and the software for submitting
data online (hemovigilance) are already in place. Fourteen
sensitization workshops/CMEs have been organized in
different parts of the country. Seventy-nine centers have
already been issued user IDs and password. Thirty-nine
blood centers have started submitting their data online. The
first issue of the hemovigilance newsletter (January to June
2013) was launched in the first half of 2013 and the third
issue (January to June 2014) is on the website and also widely
disseminated to various medical institutions and blood
centers. Updated information is available at the website of
National Institute of Biologicals, NOIDA (www.nib.gov.in).
The success of any programme depends upon participation,
the more the data we collect; more meaningful will be data
analysis and consequent blood safety recommendations for
the country.
suggested reading
1. An Action Plan for Blood Safety. National Aids Control
Organisation, Ministry of Health and Family Welfare. Govt. of
India. 2003.
2. Bisht A, Singh S, Marwaha N. Hemovigilance program- India.
Asian J Transf Sci. 2013;7:73-4.
3. deVries RRP, Faber JC, Strengers PFW. Hemovigilance: an
effective tool for improving transfusion practice. Vox Sang.
2011;100:60-7.
4. Engelfreit CP, Reesink HW. Hemovigilance systems. Vox Sang.
1999;77:110-20.
5. Faber JC. Heamovigilance in Europe: the European Heamo­
vigilance network. Transf Clin Biol. 2001;8:285-90.
6. Hemovigilance Module, Biovigilance component. National
Healthcare Safety Network. Centers for Disease Control and
Prevention USA. http://www.cdc.gov/nhsn/PDFs/hemovig
ModuleProtocol_current.pdf (last accessed in Nov 2013).
7. Hemovigilance Programme of India. National Institute
of Biologicals and Indian Pharmacopoeia Commission
 Chapter 69  Hemovigilance and its Launch in India  263
Collaboration. http://www.nib.gov.in/hemovigilance.html
(last accessed in Dec 2013).
8. International Hemovigilance Network. http://www.ihn-org.
com (last accessed on Jan 8, 2014).
9. International Society of Blood Transfusion Working Party on
Hemovigilance. Proposed standard definitions for surveillance
of non infectious adverse transfusion reactions. July 2011.
http://www.isbtweb.org/Hemovigilance/ISBT_definitions_
final_2011 (last accessed on Jan 8, 2014).
10. Pharmacovigilance programme of India for Assuring Drug
Safety http://www.cdsco.nic.in/pharmacovigilance_intro.htm
(last accessed on Jan 8, 2014).
11. Rouger Ph, Noizat-Pirena F, Le Pennac PY. Hemovigilance and
Transfusion Safety in France. Vox Sang. 2000;78:287-9.
12. Serious Hazards of Transfusion (SHOT) UK. SHOT
organization. http://www.shotuk.org.
13. Sharma RR, Kumar S, Agnihotri SK. Sources of preventable
errors related to transfusion. Vox Sang. 2001;81:37-41.
14. Todd A. Hemovigilance – closing the loop. Vox Sang. 2002;83
(Suppl I):13-6.
15. Transfusion and Transplantation Reactions in Patients. Dutch
Hemovigilance system. http://www.tripnet.nl (last accessed
on Jan 8, 2014).
16. Williamson L, Cohen H, Love E, Jones H, Todd A, Soldan K.
The Serious Hazards of Transfusion (SHOT) Initiative: The UK
approach to Hemovigilance. Vox Sang. 2000;78:291-5.
17. World Health Organisation Draft Guidelines for Adverse
Event Reporting and Learning Systems. http://www.who.
int/patientsafety/events/05/Reporting_Guidelines.pdf (last
accessed in Dec 2013).
Advances in Serological Assays
for Screening Blood Donors for
Infectious Agents
Paramjit Kaur
introduction
The first infectious disease test performed on blood donations
in the United States was a test for syphilis and screening was
introduced in the 1940s. The first assays for hepatitis B surface
antigens were introduced in the early 1970s. Early 1980s
brought attention and resources to the prevention of more
serious consequences of blood transfusion when potential
transmission of acquired immunodeficiency syndrome
(AIDS) by blood transfusion was discovered. With the
evolution of new screening tests, improved donor screening
procedures and quality systems ushered in. In addition
multiple layers of safety became part of the regulatory
framework to ensure safer blood components.1,2
The frequency with which an infection is transmitted to
blood recipients depends upon the length of the asymptomatic
blood borne period, how often blood is donated during this
period and the immune status of the recipient population.
The efficacy of blood donor screening test relies not only on
the ability to detect an individual infected by transfusion
transmissible infection but also on the duration of the
window period. The sensitivity of a screening assay depends
on its ability to close the window period.
The main types of assay used for blood screening are:
Immunoassays (IAs):
• Enzyme immunoassays (EIAs)
• Chemiluminescent immunoassays (CLIAs)
• Hemagglutination (HA)/particle agglutination (PA) assays
• Rapid immunochromatographic assays
• Nucleic acid amplification technology (NAT) assays.
Appropriate evaluation is required in selecting the type of
assay for each TTI, based on critical assay characteristics, such
as sensitivity and specificity, positive and negative predictive
value as well as cost and ease of use. The epidemiology and
geographical distribution of the agents are also critical.
Serological versus Molecular Assays
Serological assays for antibodies detect antibodies against
the infectious agent generated by the immune system of
70
the infected individual. Serological assays for antigens use
specific antibodies to detect constituents of the infectious
agent (e.g. HBsAg and HIV-1 p24 Ag). Molecular assays
(nucleic acid amplification tests or NAT) detect the nucleic
acid sequences, RNA or DNA of the infectious agent. They
are effective for the detection of recent infections or incident
cases while serologic assays for antibodies are effective for
the detection of prevalent cases. Improvements in technology
have brought sensitivity of newer generation serologic assays
for antibodies and combined antigen antibody assays closer
to NAT.3,4
Evolution of Hepatitis C Virus Assays
The initial clone discovered was from the NS4 region and the
derived protein was designated 5-1-1. It was expanded to form
c100-3 antigen that formed the basis of the first-generation
anti-HCV assay. Second generation assays included the c22
core antigen and the c33c antigen from the NS3 region. Third-
generation assay further included a protein from NS5 region.
Even with improved HCV antibody assays the infectious
window period remained around 66 days. HCV core antigen
EIAs shortened the window period considerably, but high cost
was the major limitation. A fourth-generation HCV antigen
and antibody assay (combination EIA) further improved
testing as two infectious markers of HCV are detected in the
same assay. Serology based assays may detect presence of
anti-HCV antibodies, HCV antigen or both simultaneously.
The testing platforms could be enzyme immunoassays
(EIAs), chemiluminescence (CLIA) or rapid tests. The CLIA
end point detection signal has better sensitivity than enzyme
based assays. The improvements in assay performance have
been termed as “generations” of the assays. In many blood
centers located within resource-poor countries the laboratory
facilities are limited and rapid tests provide an alternate
screening methodology to EIAs. They are based upon any one
of the following principles; agglutination, immunofiltration
or immuno-chromatography. The antigens used in the test
are usually same as those incorporated in third generation
EIAs. Each test strip or cassette has an inbuilt control band
 Chapter 70  Advances in Serological Assays for Screening Blood Donors for Infectious Agents  265
for validation. The reported sensitivities with these tests
ranged from 98 to 100%.5
Signal-to-cut–off ratios for commercially available assays:
CDC has recommended that a person be considered to
have serologic evidence of HCV infection only after an anti-
HCV screening-test-positive result has been verified by a
more specific serologic test (e.g. RIBA) or a nucleic acid test
(NAT). This more specific, supplemental testing is necessary,
particularly in populations with a lower prevalence of disease,
to identify and exclude false positive screening test results.
However, currently, the majority of laboratories report
positive anti-HCV results based on a positive screening assay
alone.6
The recommended anti-HCV testing algorithm has been
expanded to include an option that uses the signal-to-
cut–off (s/co) ratios of screening-test-positive results. This
can serve as an alternative to a supplemental test in some
circumstances, minimizing the number of specimens that
require supplemental testing and providing a result that has
a high probability of reflecting the person's true antibody
status.7
Signal-to-cut–off ratios are calculated by dividing the
optical density (OD) value of the sample being tested by the
OD value of the assay cut-off for that run. Analysis of enzyme
immunoassay and chemiluminescence assay data indicates
that s/co ratios can be used to predict supplemental test-
positive results. A specific s/co ratio can be identified for each
test that would predict a true antibody-positive result (as
defined by the results of supplemental testing) ≥95% of the
time, regardless of the anti-HCV prevalence or characteristics
of the population being tested.
Evolution of Human Immunodeficiency
Virus Assays
The first anti-HIV screening assays, used in the mid1980s,
were based on HIV-1 lysates. The replacement of native
viral proteins by recombinant and synthetic peptide-based
antigens, and the development of class-specific antibody-
capture and antigen-sandwich assays, resulted in reducing
the infectious window period by 20–30 days. Ag/Ab assays
present, however, some limitations. False-negative results
have been observed in primary HIV infection when Ag
declines and IgG Ab are not yet detectable. While HCV
Ag/Ab assays, based on an indirect IA format for Ab detection,
are subjected to this second ‘window phase’, this failure is no
longer observed with the most recent HIV Ag/Ab assays due
to their format based on sandwich detection of antibodies
including IgM. HIV Ag/Ab assays exhibit an overall high
sensitivity in the detection of Ab irrespective of the genotype,
including HIV-2.4
HIV assays have evolved over the last decade to produce
tests which possess novel characteristics and which can
address those diagnostic issues which remain, e.g. detection
of early infection, indeterminate results, infection in the
newborn, the need for rapid results, etc. The availability of
a fourth-generation test capable of offering an increase in
sensitivity over other antibody tests is yet another evolutionary
step. The concept of simultaneous antigen and antibody
detection is of great importance, as there is a definite need to
continue testing for early infection and to apply cost-saving
strategies.
Evolution of Hepatitis B Virus Assays
Serologic testing for hepatitis B virus (HBV) surface antigen
(HBsAg) and antibody to HBV core antigen (anti-HBc) has
historically been the foundation of blood screening, while HBV
nucleic acid testing (NAT) was recently developed to detect
HBsAg-negative, anti-HBc-negative blood units donated
during early acute infection. Even single-sample HBV NAT
may not substitute for anti-HBc screening, as indicated by
studies of donors with isolated anti-HBc who have extremely
low DNA levels undetectable by standard single-sample NAT
and who have been associated with transfusion-transmitted
HBV. Moreover, HBsAg testing may still be needed even in
the setting of combined anti-HBc and NAT screening. HBsAg-
positive units from donors in the chronic stage of infection
may contain very low or intermittently detectable DNA levels
that single-sample NAT would miss.8,9
Assays may utilize single or multiple monoclonal antibodies
and/or polyclonal antibodies to capture and detect antigen
in a variety of formats including enzyme immunoassays
(EIAs), agglutination methods, and microparticle assays with
fluorescent or chemiluminescent detection systems. Assay
platforms range from manual to fully automated, random
access, high throughput systems.
Sensitivity of HBsAg assays is most often expressed in
nano-grams (ng) of surface antigen protein per milliliter.
However, because of the complex nature of HBsAg molecules
and specificity differences in antibodies used to construct
HBsAg immunoassays and calibrate reference samples,
HBsAg standards vary in their definition of the HBsAg
ng/mL, making it difficult to compare assay sensitivity
performance unless the same standards are used. WHO has
developed an international reference standard for HBsAg
with concentration expressed as IU/mL, and although factors
to convert IU to ng were determined, the WHO reference
standard committee did not formally adopt a conversion
factor. The most sensitive assays detect HBsAg levels
≤0.1 ng/mL, but significantly less sensitive methods with
detection limits >1 ng/mL continue to be used in some
countries.8
Syphilis Serology
There are two different types of serologic assays for syphilis:
1. Nontreponemal assays, such as the rapid plasma reagin
(RPR) test, the venereal disease research laboratory
(VDRL) test, and the automated reagin test (ART), are
266  Section 3  Blood Safety
nonspecific tests that detect “reagin” antibodies directed
against an antigen called cardiolipin. Persons with active or
recently treated syphilis infections generally have reactive
results in nontreponemal tests, while uninfected persons
or persons successfully treated years earlier usually have
nonreactive nontreponemal test results, but retain specific
antibody reactivity.
2. Treponemal assays include chemiluminescence immuno­
assays, enzyme immunoassays (EIA), fluorescent trepo­
nemal antibody “absorbed” assays (FTA-ABS), Treponema
pallidum microhemagglutination assays (MHA-TPA)
and Treponema pallidum particle agglutination assays
(TP-PA). Treponemal assays test for antibodies to
antigens that are specific to treponemes. Although
treponemal assays are useful in identifying recent and
historically remote syphilis infections, they are not useful
in monitoring the progression of syphilis or response to
antibiotic therapy. Establishments that screen donors
using a nontreponemal assay as the test of record may
also use a treponemal diagnostic test for the purposes
of donor counseling or consideration of requalification
after deferral, and also for demonstrating that the reactive
screening test is a biological false positive.10
Malaria
Various test kits are available to detect antigens derived
from malaria parasites. Such immunologic (“immuno­
chromatographic”) tests most often use a dipstick or
cassette format, and provide results in 2–15 minutes. These
“Rapid Diagnostic Tests” (RDTs) offer a useful alternative
to microscopy in situations where reliable microscopic
diagnosis is not available. The detection of malarial antigen
was originally intended as a more rapid and objective
alternative to direct microscopy. However, although rapid
and reasonably objective, their sensitivity is not sufficient for
use in a screening context, as their overall sensitivity is still
less than that of blood films. The assays are generally based
on the detection of major specific proteins—histidine rich
protein 2 (HRP-2), plasmodial lactate dehydrogenase (pLDH)
or aldolase—using various rapid immunochromatographic
formats, and using whole blood as the sample of choice.11
Malaria RDTs are currently used in some clinical settings
and programs. However, before malaria RDTs can be widely
adopted, several issues remain to be addressed, including
improving their accuracy; lowering their cost; and ensuring
their adequate performance under adverse field conditions.
Serology detects antibodies against malaria parasites, using
either indirect immunofluorescence (IFA) or enzyme-
linked immunosorbent assay (ELISA). Serology does not
detect current infection but rather measures past exposure.
However, antibody detection is of limited role in endemic
areas and may lead to high donor deferral.
References
1. AABB Technical Manual 17th edition.
2. Bihl F, Castelli D, Marincola F, et al. Transfusion transmitted
infections. Journal of translational medicine. 2007.
3. Bianco C. Current concepts in transfusion transmitted diseases.
ISBT Science Series. 2011;6:56-60.
4. Bianco C. Testing strategies looking forward. ISBT Science
Series. 2010;5:230-3.
5. Marwaha N, Sachdev S. Current testing strategies for hepatitis
C virus infection in blood donors and the way forward. World J
Gastroenterol. 2014;20:2948-54.
6. CDC. Guidelines for laboratory testing and result reporting of
antibody to hepatitis C virus. MMWR. 2003;52(No. RR–3).
7. Gupta E, Bajpai M, Choudhary A. Hepatitis C virus: Screening,
diagnosis, and interpretation of laboratory assays. Asian J
Transfus Sci. 2014;8:19-25.
8. Kuhns MC, Busch Mp. New strategies for blood donor screening
for hepatitis B virus. Nucleic acid testing versus immunoassay
methods. Mol Diag Ther. 2006;10:77-91.
9. Schmidt M, Seifried E. Current concepts in serological testing
TTID. ISBT Science Series. 2011;6:61-6.
10. Sena AC, White BL, Sparling PF. Novel Treponema Pallidum
serologic tests: A paradigm shift in syphilis screening for the
21st century. Clinical infectious diseases. 2010;51(6):700-8.
11. Kitchen AD, Chiodini PL. Malaria and blood transfusion. Vox
Sanguinis. 2006;90:77-84.
 Section

4
Miscellaneous
The Evidence-based
Transfusion Medicine
Kulbir Kaur
Introduction
Evidence-based medicine (EBM) is the process of systemati-
cally reviewing, appraising and using clinical research find-
ings to aid the delivery of optimum clinical care to patients.1
Evidence-based medicine forms part of the multifaceted
process of assuring clinical effectiveness, the main elements
of which are (Fig. 1):1
• Production of evidence through research and scientific
review
• Production and dissemination of evidence-based clinical
guidelines
• Implementation of evidence-based, cost-effective practice
through education and management of change
• Evaluation of compliance with agreed practice guidance
through clinical audit and outcomes-focused incentives.
Evidence based transfusion medicine (EBTM) is defined as
judicious and explicit use of presently best available research
evidence in making decisions about the transfusion of blood
and blood components to individual patients after estimating
the risk of benefit and harm on the basis of research on
population samples, to inform clinical decision-making
for prescribing blood or blood components to individual
patients and to formulate guidelines for their application
from individual patients to health care services in general and
to extend to allied health professions.
Considered as a gift of life, blood transfusion is a vital
component of health care delivery system world over, but
the decision to transfuse blood or blood components is
taken under different circumstances. It involves many issues
related to blood donors as well as recipients of blood based
on clinical diagnosis, laboratory investigations, local and
national policies in various health care settings. Sometimes
the decision for blood transfusion has to be taken in
emergency situations while in routine cases, time permits to
make appropriate decisions and follow systematic approach,
but in any given situation the decision-making process should
be guided by the principles of evidence-based transfusion
71
medicine and in the recent past efforts have been made to
evaluate transfusion therapy based on:2
• Randomized controlled trials and their critical appraisal to
optimize transfusion practice
• Systematic reviews of assessment of the impact of blood
transfusion on patient outcomes
• Development of clinical transfusion practice guidelines
• Implementation of clinical practice guidelines.
Presently most of the available data shows that current
transfusion practices in clinical medicine are either sub-
optimal, variable and without clear cut guidelines especially
in developing countries. These variations may be due to lack
of availability of well-defined guidelines for blood transfusion
in clinical practice, lack of awareness of available guidelines
or reluctance to follow the available guidelines. Reluctance
to implement them may be at least partly due to perceived
weaknesses in the evidence base for their recommendations.
It has been proved beyond doubt that blood transfusion
is a treatment with substantial risks, associated costs and
limited supplies.3 Despite careful donor selection and the
development of highly specific and sensitive techniques
of testing, the achievement of zero risk blood still remains
an elusive goal. It is important to follow evidence based
transfusion practices because transfusion of blood/blood
Figure 1  Elements of the evidence-based transfusion medicine
270  Section 4  Miscellaneous
components are sometimes associated with adverse effects
like hemolytic transfusion reaction (HTR), transfusion
related acute lung injury (TRALI), anaphylactic reaction,
sepsis due to bacterial contamination, febrile nonhemolytic
transfusion reaction (FNHTR), transfusion associated
circulatory overload (TACO), transmission of infections like
HIV, HBV, HCV, syphilis, malaria, etc. Some studies have also
concluded that transfusion may be an independent risk factor
for increased morbidity, mortality and length of stay in blood/
blood component recipients.4
As such, most of the research in transfusion medicine has
been carried out to collect evidence and evaluate whether it is
beneficial to follow restrictive or liberal transfusion practices,
and the analysis of these results has aided in framing certain
guidelines with respect to utilization of blood and blood
components in order to optimize transfusion outcomes and
resource utilization. Research has also helped in establishing
the mechanisms related to many of the adverse transfusion
events and this in turn has facilitated in devising newer
methodologies to make blood/blood component transfusions
safer. Some of the evidence based guidelines with respect to
transfusion practices have been discussed below:
Evidence-based Red Blood Cells
Utilization Guidelines
Red cell transfusion is an extremely common treatment
throughout the world. Blood is administered to improve
oxygen delivery. An American Society of Anesthesiologists
Task Force makes the following recommendations:5
• Transfusion is rarely indicated when the hemoglobin level
is above 10 g/dL and is almost always indicated in patients
when the hemoglobin level is below 6 g/dL.
• The determination of transfusion in patients whose
hemoglobin level is 6–10 g/dL should be based on any
ongoing indication of organ ischemia, the rate and
magnitude of any potential or actual bleeding, the patient’s
intravascular volume status and risk of complications due
to inadequate oxygenation.
• The use of alternative measures to reduce allogeneic
red cell use should be considered, including pre­
operative autologous donation, intraoperative and post-
operative autologous blood recovery, acute normovo­
lemic hemodilution, and operative and pharmacologic
strategies.
• The same considerations regarding individualization of
red cell transfusions apply to critical care as perioperative
patients. Up to 40%
of the blood volume in a bleeding,
otherwise healthy young adult can be replaced with
crystalloid without the need for red cell transfusion.
Transfusion triggers for red cells in critical care must
be customized on the basis
of individual patient
characteristics.6 Clinical judgment is needed to estimate
how much more bleeding may occur and how much lower
the hemoglobin level will decrease, and then perform
expectant transfusion.7
• In the case of neonates and critically ill children, recent
randomized studies, e.g. premature infants in need of
transfusion (PINT) study support the use of a restrictive
strategy compared to more liberal criteria in case of
neonates and critically ill children. Table 1 shows the
general guidelines for transfusion to infants.8
Evidence-Based Platelet Utilization
Guidelines
Platelet transfusions are used to treat bleeding due to
critically decreased circulating platelet counts or functionally
abnormal platelets. Platelets may be used prophylactically
to prevent bleeding at prespecified low platelet counts.10
However, platelet transfusions are commonly implicated in
adverse effects like allergic/anaphylactic reactions, FNHTRs,
transmission of bacterial infection and development of
platelet refractoriness. Guidelines recommend that:10
• In general, maintain platelet count above 10,000/mm3 in
stable, nonbleeding patients.
• Platelet count to be maintained above 20,000/ mm3 in
unstable nonbleeding patients.
• Maintain platelet count above 50,000/mm3 in patients
undergoing invasive procedures or actively bleeding.
• In the case of critically ill or those undergoing
massive transfusion, a transfusion target of more than
50,000/mm3 is recommended for acutely bleeding patients
and above 100,000/mm3 for those with multiple trauma or
CNS injury. The platelet count may fall below 50,000/mm3
when more than 1.5–2 blood volumes have been replaced
with red cells. In the presence of micro vascular bleeding,
transfusion may be appropriate when counts are known or
suspected to be below 100,000/mm3.
• In case of neonates undergoing invasive procedures/minor
surgery or experiencing clinically significant bleeding may
be transfused at less than 50,000/mm3. For major surgery
or bleeding in the face of additional hemostatic stressors
(e.g. disseminated intravascular coagulation, necrotizing
Table 1: General guidelines transfusion to infants9
Maintain HCT between Clinical status
Severe cardiopulmonary disease
(e.g.
40–45%
mechanical ventilation >0.35 FiO2)
Moderate cardiopulmonary disease (e.g.
30–35% less intensive assisted ventilation such as
nasal CPAP or supplemental oxygen)
30–35% Major surgery
Stable anemia, especially if unexplained
20–30% breathing disorder or unexplained poor
growth
 Chapter 71  The Evidence-based Transfusion Medicine  271
enterocolitis) transfusion is appropriate at counts below
100,000/mm3.
• Platelets should not be used in patients with autoimmune
thrombocytopenia or thrombotic thrombocytopenic
purpura (TTP) except for life-threatening hemorrhage.
Evidence-Based Fresh Frozen Plasma
Utilization Guidelines
Fresh frozen plasma (FFP) may be among the most “high risk”
of all blood components11,12 and FFP transfusion may lead to
adverse events like TRALI, transfusion-transmitted infection,
fluid overload and allergic complications.13,14 Various studies
have shown an association between cases of TRALI and
plasma from female donors and therefore, male donors are
now preferred for production of FFP.15,16 Considering the
risks associated with FFP transfusion, a prophylactic policy
is justified only if the risk of bleeding is greater than the risk
of associated adverse effects. At present, FFP transfusion is
indicated for use in patients with the following conditions:17-22
• Active bleeding, or before surgery or an invasive
procedure in patients (adults and neonates) with acquired
deficiencies of one or more coagulation factors as
demonstrated by an increased international normalized
ratio (INR), prothrombin time (PT), or activated partial
thromboplastin time (aPTT), when no alternative
therapies are available or appropriate.
• Immediate correction of vitamin K deficiency or reversal of
warfarin effect in a patient with active bleeding, or before
surgery or an invasive procedure (in conjunction with use
of prothrombin complex concentrates).
• DIC or consumptive coagulopathy with active bleeding.
• Massive transfusion with coagulopathic bleeding.
• Thrombotic thrombocytopenic purpura (TTP).
• Active bleeding, or before surgery or an invasive procedure
in patients with a congenital factor deficiency when no
alternative therapies are available or appropriate.
• Previous common uses of FFP that are now considered
inappropriate include: volume replacement, correction
of hypoalbuminemia, nutritional support, and immuno­
globulin replacement.
Evidence-Based Cryoprecipitate
Utilization Guidelines
Cryoprecipitate is indicated for bleeding associated with
fibrinogen deficiencies and Factor XIII deficiency. It
predominantly contains only Factor VIII, vWF, fibronectin,
Factor XIII and fibrinogen. Therefore, cryoprecipitate is not
a source of all coagulation factors and, this not appropriate
replacement therapy in patients with global coagulation
factor deficiencies (e.g. with liver disease). Its use should be
reserved for:
• Patients with hemophilia A or von Willebrand’s
disease
(vWD) should only be treated with cryoprecipitate when
appropriate Factor VIII concentrates or Factor VIII
concentrates containing FVIII: vWF are not available.
• Do not transfuse cryoprecipitate unless laboratory studies
confirm deficiency of a specific clotting protein for which
this component is indicated (e.g. fibrinogen).23
• In case of critically ill patients, with Hypofibrinogenemia/
dysfibrinogenemia, transfuse for bleeding associated with
fibrinogen levels less than 100 to 120 mg/dL or reduced
functional levels of fibrinogen.24
• Cryoprecipitate is especially useful when it is not possible
to give enough FFP to provide adequate levels of fibrinogen
without volume overloading the patient.
• Cryoprecipitate has been used for uremic bleeding
but
efficacy has not been clearly demonstrated, and
1-deamino-8-D-arginine vasopressin (DDAVP) and other
modalities are preferred.25
Limitations of Evidence-Based
Transfusion Medicine
Despite the emphasis on following evidence-based practices,
evidence based medicine too has certain limitations.
• EBM alone cannot provide a clinical decision. Each deci-
sion will also be based on the individual patient factors,
available resources, clinical expertise and the costs of the
intervention.
• Patients enrolled in clinical trials are not always
representative of individual patients requiring treatment
and generalizing to different clinical settings may not be
appropriate.
• In EBM there is over emphasis on methodology at the
expense of clinical relevance.
• Evidence base for much of the practice has not developed
to the point at which it can be universally applied with
confidence.
• Even when clear-cut evidence based guidelines are avail-
able, there may be a lack of compliance and dissemina-
tion.26
Some of these limitations can be overcome by increasing
sharing of expertise and resources between local, national
and international scientific communities and by avoiding
the duplication of research, increasing opportunities to dis-
seminate the work that 
has already been done and planning
future research.2
Conclusion
Research shows that many a times inappropriate transfusion
practices are followed which may not provide any substantial
benefits but on the contrary make the patient prone to the
potential adverse effects that are sometimes associated
with blood transfusion. Therefore, it is essential to identify
those patients who are at risk of complications if devoid of
transfusion, and to avoid unnecessary transfusions.3 Effective
implementation of evidence based transfusion guidelines
272  Section 4  Miscellaneous
combined with continuous feedback and research will 12. Khan H, Belsher J, Yilmaz M, et al. Fresh-frozen plasma
facilitate in devising newer approaches to make blood/blood and platelet transfusions are associated with development
component transfusions safer. of acute lung injury in critically ill medical patients. Chest.
2007;131:1308-14.
13. Holness L, Knippen MA, Simmons L, Lachenbruch PA.
References Fatalities caused by TRALI. Transfus Med Rev. 2004;18:184-8.
14. Andrzejewski C, Popovsky MA. Transfusion-associated adverse
1. Belsey J. http://www.medicine.ox.ac.uk/bandolier/painres/
pulmonary sequelae: Widening our Perspective. Transfusion.
download/whatis/ebm.pdf.
2005;45:1048-50.
2. Murphy MF, Brunskill S, Estcourt L, Stanworth S, Dorée C. How
15. Serious hazards of transfusion. Annual report 2005. Manchester,
to further develop the evidence base for Transfusion Medicine.
UK: SHOT Office, 2005. [Available at http://www.shotuk.org.].
Blood Transfusion. 2012;10:436-9.
16. Clarifications to recommendations to reduce the risk of TRALI.
3. Shander A, Gross I, Hill S, Javidroozi M, Sledge S. New
Association bulletin #07-03 (November 28, 2007). Bethesda,
perspective on best transfusion practices. Blood Transfus.
MD: AABB, 2007.
DOI 10.2450/2012.0195-12.
17. Practice parameter for the use of fresh-frozen plasma, cryopre-
4. Makroo RN, Walia RS, Aneja S, Bhatia A, Chowdhry M.
cipitate and platelets. Fresh Frozen Plasma, Cryoprecipitate
Preoperative predictors of blood component transfusion in
and Platelets Administration Practice Guidelines Development
living donor liver transplantation. Asian J Transfus Sci. 2013;7(2):
Task Force of the College of American Pathologists. JAMA.
140-6.
1994;271:777-81.
5. Anonymous. Practice guidelines for perioperative blood
18. Canadian Medical Association Expert Working Group.
transfusion
and adjuvant therapies: an updated report by
Guidelines for red blood cell and plasma transfusion for adults
the American Society of Anesthesiologists Task Force on
and children. Can Med Assoc J. 1997;156(11 suppl):S1-24.
Perioperative Blood Transfusion and Adjuvant Therapies.
19. Roseff SD, Luban NLC, Manno CS. Guidelines for assessing
Anesthesiology. 2006;105:198-208.
appropriateness of pediatric transfusion. Transfusion. 2002;42:
6. Hebert PC, Wells G, Blajchman MA, et al. A multicenter,
1398-413.
randomized, controlled clinical trial of transfusion require­
20. Contreras M, Ala FA, Greaves M, et al. Guidelines for the use
ments in critical care. Transfusion Requirements in Critical
of fresh frozen plasma. British Committee for Standards in
Care Investigators, Canadian Critical Care Trials Group. N Engl
Haematology, Working Party of the Blood Transfusion Task
J Med. 1999;340:409-17.
Force. Transfus Med. 1992;2:57-63.
7. Carson JL, Hebert P. Anemia and Red Blood Cell Transfusion.
21. NIH consensus conference. Fresh-frozen plasma. Indications
Simon TL, Snyder EL, Solheim BJ, Stowell CP, Strauss RG,
and risks. JAMA. 1985;253:551-3.
Petrides M (Eds). Rossi’s Principles of Transfusion Medicine.
22. Practice guidelines for blood component therapy: A report by
4th edn. Wiley-Blackwell. 2009. pp.131-48.
the American Society of Anesthesiologists Task Force on Blood
8. Kirpalani H, Whyte RK, Andersen C, et al. The premature
Component Therapy. Anesthesiology. 1996;84:732-47.
infants in need of transfusion (PINT) study: A randomized,
23. Circular of Information for the Use of Human Blood and Blood
controlled trial of a restrictive (low) versus liberal (high)
Components. Prepared jointly by the AABB, America’s Blood
transfusion threshold for extremely low birth weight infants.
Centers and the American Red Cross. July 2002.
J Pediatr. 2006;149:301-7.
24. Bethesda, MD. Guidelines for blood utilization review.
9. Strauss R. RBC transfusion and/or recombinant EPO therapy of
American Association of Blood Banks, 2001.
the anaemia of prematurity. ISBT Science Series. 2006;1:11-4.
25. Mannucci PM. How I treat patients with von Willebrand
10. Miller Y, et al. Practice Guidelines for Blood Transfusion:
disease. Blood. 2001;97:1915-9.
A Compilation from Recent Peer-Reviewed Literature
26. Murphy MF, Pamphilon DH (Eds). Getting the most out of
2ndedition American Red Cross 2007. http://www.redcross.
evidence for transfusion medicine. Practical Transfusion
org/wwwfiles/Documents/WorkingWiththeRedCross/
Medicine. 3rd edn, chapter 47. Wiley-Blackwell.
practiceguidelinesforbloodtrans.pdf.
11. MacLennan S, Williamson LM. Risks of fresh frozen plasma
and platelets. J Trauma. 2006;60:S46-50.
Blood Bank Accreditation
BK Rana
Keywords: Accreditation; Blood bank; Blood centre;
Improvement; QCI: Quality Council of India; National
Accreditation Board for Hospital and Healthcare Providers;
Transfusion service.
Introduction
Blood Safety is now talking of the town. We all are aware that
blood banks/centres are regulated by Central Drugs Licensing
Authority under Drugs and Cosmetics Acts and Rules. In a
manner, blood banks/centres are fairly regulated compared
to medical laboratories; however, it is observed that it’s
not enough to ensure quality of blood and its products. To
support the licensing mechanism and ensure that blood and
its products meet the specified requirement and provide
an opportunity for BB to improve on a continuous basis,
the Quality Council of India (QCI) through one of its Board,
National Accreditation Board for Hospitals and Healthcare
Providers (NABH) has therefore introduced standards for
Blood Banks/Centres and Transfusion Services in 2007. This
chapter deals briefly with the standards structured by the
NABH for accreditation of blood banks/centres.
Speeding changing market forces, increasing health
awareness, and the entry of the corporate sector into health
delivery have raised the demand for quality in healthcare
services. Particularly, when a patient comes to know that
transfusion can be fatal instead of lifesaving. It became all
the more critical that a blood or its product must confirm
to highest quality norms set by regulator who grants license
to run a blood bank. The phenomenal growth in blood
banking and transfusion services has further enhanced the
importance of this field. Development of newer technologies
to detect and identify disease markers has made tremendous
contribution in the quality of services. NABH accreditation
for hospitals takes into account major aspects of blood
banking and transfusion services, although its role and scope
are limited. Hence, to assess the quality and safety of blood
and its products and to have a method for the monitoring
of quality standards, a basic accreditation program has
been introduced in the country. These standards reflect the
72
expectation of good services from the viewpoint of the service
provider, the patient, and the referrer, as well as the regulatory
bodies.
Types of Blood Banks/Centres
National Accreditation Board for Hospitals and Healthcare
Providers (NABH) has classified blood banks/centres on
the basis of its management (Government, Indian Red
Cross, Voluntary/Charitable or any other) and functionality
(Hospital based or standalone). It means that there are
no restrictions on any licensed blood bank for making
application for accreditation on the basis of its management
and function.
Scope of Accreditation
This standard covers whole blood and all its products
including molecular typing, HLA typing and therapeutic
procedures. In a way, all those activities for which a license
can be granted by the licensing authority are covered.
Requirements of Accreditation Standards
NABH Accreditation Standards for Blood Banks/Centres and
Transfusion Services are divided into eleven clauses and each
clause has been further divided into several sub-clauses.
This chapter describe brief outline of these clauses and sub-
clauses.
Organization and Management
The blood bank/blood centre should be legal identity and
must have a valid license from Central Drugs Standard Control
Organization (CDSCO) and approved by Drug Controller
General (India), central license approving authority under
Drug and Cosmetic Rules-1945 with further amendments. The
blood bank is required to define its organization structure in
terms of various positions and their functions (responsibilities
and authorities). Blood bank must at all times comply with all
274  Section 4  Miscellaneous
applicable legal requirements of the country. Staff should be
bound by the ethical code of their respective profession. The
Management (a person or group of persons responsible for
managing the functions of the blood bank at which decisions
are made) must define and implement a quality system which
at a minimum meets the requirements of NABH accreditation
standards including its quality policy documented in a quality
manual. Quality policy contains scope of services, objective of
quality management system with management commitment
to comply with the standards and local regulations. Quality
manual is considered as an apex document which guides
the blood bank for its functions through policies, processes
and procedures. Staff must understand and follow the
documented system as described in the quality manual.
Management needs to identify a Quality Manager, who will
be responsible for the implementation and maintenance of
quality management system, and a Technical Manager, who
will be responsible for technical operation.
Accommodation and Environment
The premises, used for operation of a blood bank/blood
centre and/or preparation of blood components shall be
constructed in such a manner so as to permit the operation
of the blood bank/blood centre and preparation of blood
components under hygienic conditions and shall avoid entry
of insects, rodents and flies. It should be well-lighted and
ventilated. The blood bank/blood centre shall be designed for
the efficiency of its operation, to optimize the comfort of its
occupants and to minimize the risk of injury and occupational
illness. Patient/recipients, employees and visitors should
be protected from recognized hazards. A blood bank/blood
centre shall have a minimum area as defined by licensing
authority for its operations. It should provide necessary space
for registration and medical examination, blood collection,
refreshment-cum-rest-room, laboratory for testing of blood
transmissible disease markers like hepatitis, syphilis, malaria,
HIV-antibodies, blood component preparation (if applicable),
sterilization-cum-washing, and store-cum-record room. All
these facilities must have effective separation between in
order to avoid incompatible activities. Access to areas affecting
the quality of the examinations should be controlled. A good
housekeeping practice must be followed. It should have
adequate back up facility for maintaining electrical supply
round the clock for effective operation. The blood bank/
blood centre should have process to minimize and respond
to environmentally related risks to the health and safety of
employees (including immunization), donors, volunteers,
patient/recipients and visitors. Suitable measures should be
taken for monitoring of adherence to biological, chemical and
radiation safety standards and regulation, where applicable.
Proper communication facilities for communication within
and outside the center must be available.
Personnel
The blood bank/blood centre is required to employ an adequate
number of individuals qualified by education, training
and experience. The operation of blood bank/blood centre
should be conducted under the active direction and personal
supervision of competent technical staff. Qualification and
experience for Director/In-charge/ medical officer/in-charge,
supervisors, technicians and nurses must be at the minimum
as required by licensing authority. Each staff must be given
a documented job description to ensure that staff Director/
In-charge understands their responsibilities and authority to
discharge their duties appropriately. All personnel employed
should also have training specific to quality assurance and
quality management. It is the responsibility of the management
to ensure that all employees are trained to prevent adverse
incidents and/or contain the effects of, and report adverse
incidents. Blood Centre should assess the competency of
each staff to perform assigned tasks following training and
periodically thereafter. Retraining and reassessment must
be provided based on competency analysis, when necessary.
A pre-employment medical examination and regular
health checkup must be conducted on all the employees.
It is required not only to protect the employees from deadly
infection but also safeguard the blood centre from certain
legal implications. Blood centre must maintain records of the
personal information, relevant educational and professional
qualification, training and experience, and competence of
all personnel. Staffs has to strictly maintain confidentiality of
information regarding donor/patient/recipient.
Equipment
The blood centre must be in the possession of all the equip­
ment those are required for blood collection, component
preparation, processing, examination and storage, as
appropriate as per the scope of license. It is must that equipment
are properly calibrated, maintained, and monitored for their
functioning and kept in a clean and proper manner and so
placed as to facilitate regular cleaning and maintenance.
To protect the equipment from damage and improper use,
only authorized personnel should operate them. Up-to-date
instructions on the use and maintenance of the equipment
(including relevant manuals and direction for use provided by
the manufacturer of the equipment) shall be readily available
to personnel. It is essential that certain records related to
equipment are maintained. These may be but not limited to
manufacturer’s name, type, identification and serial number
or other unique identification, manu­facturer’s contact person
and telephone number, date of receiving and date of putting
into a service, current location, where appropriate, condition
when received (new, used or reconditioned), manufacturer’s
instructions, if available, or reference of their retention,

equipment performance records that confirm the equipment
suitability for use, maintenance carried out and that planned
for the future, damage to or malfunction, modification or
repair of the equipment. Blood centre is required to establish
a programme that regularly monitors and demonstrates
proper calibration and function of instruments, reagents
and analytical system. It shall also have a documented and
recorded programme of preventive maintenance, which, at
a minimum, follows the manufacturer’s recommendation.
Calibration of equipment is essential to ensure correct results
and therefore these must be calibrated regularly on scheduled
basis as described in the standard operating procedure for
that particular equipment. Calibration can be done in-house
as well as by outside agency using master calibrators. As
per policy, which NABH is intend to follow soon equipment
must be either calibrated in-house using proper procedures,
conditions and master calibrators or by an NABL accredited
calibration laboratory in that specific field. This is to ensure
unbroken chain of traceability of measurements to national/
international standards.
External Services and Supplies
There must be documented policies and procedures for the
selection and use of purchased external services, equipment
and consumable supplies that affect the quality of its
services. Purchased items shall consistently meet the quality
requirements. There should be procedures and criteria
for inspection and acceptance/rejection of consumable
materials. All supplies and reagents used in the collection,
processing, compatibility testing, storage and distribution
of blood and blood components shall be stored at proper
temperature in a safe and hygienic place. A proper inventory
control system should be in place. This system should include
the recording of lot number of all relevant reagents, control
materials and calibrators, the date of receipt in the center
and the date the material was placed in service. Evaluation
of suppliers of critical reagents, supplies and services that
affect the quality of examinations must be done and a record
is maintained of these evaluations.
Process Control
Policies and validation of processes and procedures: The blood
bank/blood centre is required to have policies, processes and
procedures to ensure the quality of the blood, component,
derivatives and services, and ensure that these policies,
processes and procedures are carried out under controlled
conditions. There should be a mechanism to identify who
performed each critical step in collection, processing,
compatibility testing and transportation of blood, component
and derivatives issued, and when it was performed. It is the
responsibility of blood bank/blood centre to ensure that all
blood, components, derivatives issued and critical materials
Chapter 72  Blood Bank Accreditation  275
used in their processing activities, as well as laboratory sample
and donor and patient/recipient records, are identified and
traceable. In case, donor who is subsequently found to have
been infected with transfusion transmissible infection, the
blood bank/blood centre shall inform the patient/recipient’s
physician. Appropriate record of such events shall be kept.
The unused components from this unit shall be discarded.
All procedures including examination procedures,
which meet the needs of the users of blood bank/blood
centre services, should only be used. National guidelines/
manuals and other regulatory directives shall be followed
at all times. In case, in-house procedures are used, these
shall be appropriately validated for their intended use and
fully documented. Record of the results obtained and the
procedure used for the validation in case in-house developed
methods are used, must be kept. All procedures shall be
documented and be available at the workstation for relevant
staff. It is a good practice if procedures and necessary
instructions can be made available in a language commonly
understood by the staff.
As a policy, blood bank should try to collect blood from
voluntary, nonremunerated, low risk, safe and healthy
donors. Donors are educated prior to collection of blood
regarding the risk factors of transfusion transmissible
infections and maintain a voluntary donor directory.
Pre-donation counselling by the counsellor/staff with
appropriate training shall be made available maintaining
privacy and confidentiality. A medical officer with minimum
MBBS qualification should be reviewing the donor’s health
conditions and physical examination of the donor. Prior to
blood donation, the consent of the donor shall be obtained in
writing with donor’s signature or thumb impression after the
procedure is explained and the donor is informed regarding
testing of blood for all mandatory tests for safety of recipients.
The donor shall be provided an opportunity to ask questions
and refuse consent. After donation, if the donor seeks the
status of transfusion transmitted infection (TTI), the same
may be provided with prior consent.
Blood shall be collected only by a licensed blood bank/
blood centre and shall be drawn from the donor by a
qualified physician or under his/her supervision by nurse/
technician trained in the procedure. A physician is to be
present on the premises when the blood is being collected.
Blood shall be collected by single venipuncture and flow
of blood shall be continuous. The blood bags for collection
of blood shall be sterile, pyrogen-free and disposable, with
a closed system of collection as per standards provided by
national authority. Necessary drugs and equipment shall
be available for treatment of donor reaction, if any. Donor
blood collection staff shall be trained in identification and
management of donor reactions like Syncope (fainting or
vasovagal syndrome), Tetany (twitching or muscular spasm),
nausea and vomiting, hematoma, convulsions, and cardiac
problems.
276  Section 4  Miscellaneous
Blood donation camps shall be organized only by blood
bank/blood centres authorized by State Blood Transfusion
Council (SBTC) to augment blood stocks. Adequate publicity
and information education and communication (IEC)
material shall be made available to the organisations. It is of
utmost importance that the number of blood units collected
shall commensurate with the actual requirement of blood
units rather than by social or emotional pressures.
To ensure blood safety, the blood bank/blood centres
shall provide pre- and post-donation counselling services. All
blood bank/blood centres shall train their donor organisers/
medical officers to undertake counseling in the absence of
a donor counsellor. Donors who are HIV sero-reactive shall
be referred to a voluntary counseling and testing center
(VCTC) for post donation confirmation and counselling or
may be provided with the facilities for the confirmation and
counseling at blood bank/blood centre. For TTI other than
HIV, the donor shall be referred for follow-up to concerned
speciality for further management.
In case, components (red blood cells, washed red cells,
leucocyte depleted red blood cells, Frozen and deglycerolised
red blood cell concentrate, random donor platelets,
granulocyte concentrate, single donor plasma, fresh frozen
plasma, cryo poor plasma or factor VIII deficient plasma,
single donor cryoprecipitate) are being prepared, the sterility
of all components shall be maintained during processing by
the use of aseptic methods and sterile pyrogen-free disposable
bags and solutions.
A designated area shall be used for storage to limit
deterioration and prevent damage to materials in process and
final products. The access to such areas shall be controlled.
Donated blood must be tested for ABO group, unexpected
antibodies and transfusion transmitted infection (TTI) which
include HIV, vital hepatitis, syphilis, and malaria.
Blood can only be issued by the blood bank/blood centre
along with the blood cross matching report. In case of
emergency, where delay in providing blood may jeopardize
the patient/recipient’s life, blood or blood components shall
be issued before completion of routine cross matching tests,
on receipt of a signed written request of the treating physician
stating that the clinical condition of the patient/recipient is
sufficiently urgent to require the issuance of blood before
completing ABO and Rh(D) tests and compatibility testing.
Records of such requests shall be retained for 5 years.
Transfusion reactions must be monitored and documented
and a root cause analysis must be carried out to prevent such
incidents in future.
To ensure quality, the blood bank/blood centre must
practice internal quality control measures and also participate
in external quality assurance scheme (EQAS)/proficiency
testing programme (PT).
Identification of Deviations and Adverse Events
The blood bank/blood centre is required to implement a
defined policy and procedure when any aspect of its test
analysis or function does not conform to laid down procedure.
It allows blood bank to identify deviation, if any, caused in its
operations and take necessary measures.
Performance Improvement
The blood bank/blood centre must put a policy and procedure
in place for addressing complaints, or other feed backs
received from donors, clinicians, blood camp organizers
or other parties. These can be used as improvement tools.
Record of complaints, investigations and corrective actions
taken by the blood bank/blood centre shall be maintained.
Procedure for corrective action shall include a process of
investigation to determine root cause of the problem.
Blood bank/blood centre management shall monitor
the results of any corrective action taken, in order to ensure
that they have been effective in overcoming the identified
problems. Whenever, the investigation casts doubt on
compliance with policies and procedures, blood bank/blood
centre shall ensure that an additional audit is done for that
area. All corrective actions are documented and recorded
with root cause analysis and submitted to management
review meeting. Preventive action is a proactive process for
identifying opportunities for improvement, whenever they
are identified either technical or concerning the quality
system. The action plan needs to be developed, implemented
and monitored to reduce the likelihood of the occurrence of
such nonconformities.
NABH has recently defined following ten quality indica­
tors for blood banks to monitor. Out if these ten, first five
are mandatory to monitor and report to NABH every six
months. It will help NABH and blood banks to keep an eye on
improvement being demonstrated.
Combined TTI cases
(HIV + HBV + HCV + syphilis + MP)
i. TTI% = × 100
Total no. of donors
ii. Adverse transfusion reaction rate% =
No. of adverse transfusion reactions
× 100
Total number of blood and
component issues
(All major and minor reactions to be classified as per
NHvPI and reported to blood bank)
iii. Wastage rates =
No. of blood/blood components discarded
× 100
Total no. of blood/blood components issued
iv. Turnaround time (TAT) of blood issues =
Sum of the time taken
× 100
Total number of blood and blood
components cross matched/reserved
(Time taken to be calculated from the time the request/
sample is received in the blood bank till the blood is

cross matched/reserved and available for transfusion.
Blood Bank shall set upper limits for routine and
emergency issues separately).
v. Component QC failures (for each component) =
No. of component QC failures
× 100
Total no. of component tested
vi. Adverse donor reaction rate% =
No. of donors experiencing adverse reaction
× 100
Total no. of donors
vii. Donor deferral rate% =
No. of donor deferrals
× 100
Total no. of donation + total no. of deferrals
viii. %of components =
Total component issues
× 100
Total whole blood + component issues
ix. TTI outliers %age =
No. of deviations beyond +/– 2SD
× 100
Total no. of batch assays
x. Delays in transfusion beyond 30 minutes after
issue—sample audit by BB every month.
Document Control
Blood bank/blood centre define document and maintain
procedures to control all documents and information
(from internal and external sources) that form its quality
documentation. In this context, ‘document’ is any information
or instructions, including policy statements, text books,
procedures, specifications, calibration tables, biological
reference intervals and their origins, charts, posters, notices,
memoranda, software, drawings, plans, and documents of
external origin such as regulations, standards or examination
procedures.
Records
All records relevant to the quality management system are
uniquely identified and appropriately labeled. Policies,
processes and procedures to ensure that records are
identified, reviewed, retained and that records are created,
stored, and archived in accordance with record retention
policies. A policy, that defines the length of time various
records pertaining to quality management system or technical
records are to be retained, is to be laid down.
Chapter 72  Blood Bank Accreditation  277
Internal Audit and Management Review
Management review and internal audits of all elements of the
system, both managerial and technical, is to be conducted at
regular intervals but not less than once in twelve months in
order to verify that operations continue to comply with the
requirements of the quality management system. Audits are
planned, organized, and carried out by the quality manager
or designated qualified personnel. Personnel should not
audit their own activities to prevent conflict of interest.
The procedures for internal audits need to be defined and
documented and include the types of audit, frequencies,
methodologies and required documentation. If deficiencies
or opportunities for improvement are noted, the blood bank/
blood centre must undertake appropriate corrective and/or
preventive actions. All elements of the quality system should
be covered in internal audit at least once every twelve months.
Blood bank/blood centre management is required to
review its quality management system and all of its medical
services, including examination and advisory activities, to
ensure their continuing suitability and effectiveness. The
results of such reviews shall be incorporated into a plan that
includes goals, objectives and action plans. A typical period
for conducting a management review is once every twelve
months. The results of internal audits must be a part of the
agenda. Findings and the actions that arise from management
reviews are recorded, and blood bank/blood centre is
informed of these findings and the decisions made as a result
of the review. Blood bank/blood centre management will
take necessary actions to address the concerns raised in the
meeting within an appropriate and agreed-upon-time.
Conclusion
Therefore, every blood bank must aim for ‘Zero Tolerance’
or ‘Error Free’ operations in its activities to offer better
patient care and ensure patient safety. It is nice to learn
that organisations like National AIDS Control Organisation
(NACO) and State Blood Transfusion Councils are advocating
importance of accreditation. The ISBTI and ISTM are also
playing a vital role in creating awareness amongst blood
bankers and stakeholders.
suggested reading
1. Accreditation Standards on Blood Banks/Blood Centres
and Transfusion Services, 2nd edn, June 2013: National
Accreditation Board for Hospitals and Healthcare Providers
(NABH).
2. ISO 15189: 2012- Medical Laboratories: Requirements for
Quality and Competence.

National Plasma Policy for Access
to Plasma Derived Proteins for
Clinical/Therapeutic Use
Harprit Singh, Shobini Rajan, Shanoo Mishra, Sunil D Khaparde
INTRODUCTION
The plasma policy aims at making available, easily acces-
sible and adequate supply of high quality of human plasma
derived proteins for clinical/therapeutic use. The plasma is
prepared as part of safe and quality blood and blood compo-
nents collected/procured from a voluntary non-remunerated
regular blood donor in well-equipped premises, which is free
from transfusion transmitted infections, and is stored and
transported under optimum conditions.
Plasma has limited utility in its raw form for various
coagulopathies, plasma exchange, etc. but is one such
important blood component which can be further processed
to make many more lifesaving proteins of immense clinical
significance. Such proteins are known as plasma derived
proteins (PDP). Example of PDP include albumin, coagulant
proteins such as FVIII, immunoglobulin’s such as IVIg,
Hyperimmune sera e.g. HBIg, Tetanus Ig, etc. Plasma
forms the raw material for the manufacture of plasma
derived proteins (PDP). Currently plasma derived proteins
are manufactured within the country in limited quantity
by existing Plasma Fractionation Centres. These centers
fractionate the unused plasma recovered from whole blood
at various licensed blood component separation units of the
country. Significant quantity is obtained through import from
other countries.
At present, all the recovered plasma is not being used
clinically or for plasma fractionation. The policy aims at
enabling the mobilization of this excess plasma stocks at the
blood banks for fractionation to make some more high value
products, which hitherto are not often available in adequate
quantities to meet the increasing clinical requirements.
The process of collecting standard plasma and transporting
them under optimum conditions for fractionation, identifying
critical parameters for safety, ensuring compliance with
regulatory requirements, training for the appropriate usage
of these products will be covered under this policy. The
73
policy reiterates the endeavor of the government to facilitate
supply of affordable products to the needy, regardless of their
economic status. The policy will result in a comprehensive,
efficient way to optimize usage of plasma for the manufacture
of high quality blood components, and make our country
self-reliant and standardize their availability and utilization
through comprehensive, efficient and a total quality
management approach.
OBJECTIVES OF THE POLICY1
To achieve the aim of facilitating national access to PDP for
clinical/therapeutic use, the following objectives are drawn:
• To reiterate that Government will facilitate availability and
utilization of safe and adequate quantity of plasma derived
products for clinical/therapeutic use.
• To make available adequate resources to develop and
organize the plasma/PDP mobilization throughout the
country.
• To take adequate regulatory and legislative steps for
monitoring of activities related to plasma derived products.
• To encourage Research and Development in the field
of blood components, plasma fractionation and plasma
derived products.
• To strengthen quality systems in blood transfusion
services for plasma collection, transportation, processing,
production and distribution of PDP.
Objective 1
To reiterate that Government will facilitate availability of
safe and adequate quantity of plasma derived products for
clinical/therapeutic use.
Strategy
1.1 To augment set up and functioning of blood component
separation units (BCSU) with the help of national and
 Chapter 73  National Plasma Policy for Access to Plasma Derived Proteins for Clinical/Therapeutic Use  279
state blood transfusion services in the country in order
to optimize recovery and utilization of plasma.
1.2 To evolve processes and methods to standardize
logistics of plasma collection, transport and storage
from blood banks to warehouses/Plasma Fractionation
Centres (PFC) and transport of plasma derived products
(PDP) to distribution outlets.
1.3 To put in place mechanisms to improve co-ordination
and interaction between various BCSUs and plasma
warehouses/PFCs in order to achieve desired end
product quality.
1.4 To advocate for effective and judicious clinical use of
human plasma and PDP to minimize unwarranted use
of whole blood/ plasma/PDP.
1.5 To formulate national guidelines on ‘Clinical use of
plasma derived products’ and update as required from
time to time.
1.6 To review plasma/PDP utilization by various facilities
periodically at state and national level.
1.7 To promote interdepartmental activities with all
concerned including other Ministries, stakeholders and
health programs that would help optimize production
and utilization of PDP.
1.8 To facilitate access and availability of PDP to cater to
special requirement including remote locations will be
done with closed coordination with DGAFMS.
1.9 To establish latest technology and time to time up
gradation to bring about self-sufficiency for PDP.
1.10 To participate in public private partnership/
collaborations to improve production and improve
availability of PDP.
1.11 To evolve mechanisms for periodical review and
evaluate the implementation of the policy across the
country.
Objective 2
To make available adequate resources to develop and
organize the plasma mobilization throughout the country.
Strategy
2.1 To support/strengthen the existing network of Blood
Transfusion Services (BTS) so as to consolidate
and improve blood and plasma donor base, blood
componentization and recovery of good quality of
plasma.
2.2 To allocate resources and funds in existing public health
programs as well as advocate for resource allocation by
corporate sectors, bilateral/international/agencies for
plasma mobilization.
2.3 To additionally strengthen source plasma collection
through existing BCSU/Apheresis centers within
existing network of blood transfusion services.
2.4 To ensure engagement of trained manpower at all
levels to facilitate plasma mobilization.
2.5 To ensure proper infrastructure, equipment and
transportation facilities to have high quality of plasma.
2.6 To direct efforts towards recruitment and retention of
voluntary, non-remunerated blood donors, through
education and awareness programs also incorporating
incorporate IEC strategies, NGO involvement and
special donor registries for hyperimmune products,
etc. as an integral part of voluntary blood donation
programs.
2.7 To standardize pricing of PDP, with the help of existing
policies/resources, to ensure not for profit but techno-
financial viable and self-sustaining mechanisms.
Objective 3
To take adequate regulatory and legislative steps for monitor-
ing of activities related to plasma derived products.
Strategy
3.1 To facilitate the regulatory approval of updated
methodology with the purpose of increasing plasma
recovery from donated blood.
3.2 To review the regulatory framework with respect
to availability/manufacturing and distribution of
acceptable quality of PDP for clinical use.
3.3 To review and update Standards, Drugs and Cosmetics
Act/Rules and Indian Pharmacopoeia from time to
time.
3.4 To periodically review the existing provisions of
prevailing regulatory frameworks well as introduce
stringent penalties for unauthorized/irregular practices
in plasma processing and delivery of PDP.
Objective 4
To encourage Research and Development in the field of
blood components, plasma fractionation and plasma derived
products.
Strategy
4.1 To organize capacity building/exposure visits/
hands on training of personnel dealing with plasma
fractionation, related to all process and quality aspects.
4.2 To facilitate research in blood components, plasma
fractionation and PDP in association with recognized
bodies including ICMR, DST and DCGI.
4.3 To make available financial support for the conduct
of R and D in processing of plasma and PDPs through
various channels.
4.4 To collaborate with industry and academia to launch
blood products faster and promote Inter-country and
intra-country exchange for training and experience of
personnel associated with plasma fractionation.
4.5 To direct efforts towards development of indigenous
kits/processes and technology, to make them cost
competitive.
280  Section 4  Miscellaneous
4.6 To facilitate evidence based practices in research
involving utilization of human plasma/blood, from
units unused/discarded from blood banks due to any
reason and evolve a regulatory framework thereof.
Objective 5
To strengthen Quality Systems in blood transfusion services
for plasma collection, transportation, processing, production
and distribution of PDP.
Strategy
5.1 To set national quality standards covering all aspects
in manpower, equipment, processes, procedures,
Reference
products and quality systems.
5.2 To articulate a continuous all round improvement
program in plasma fractionation as part of quality
systems as an endeavor to work towards gold standards.
5.3 To mandate that Plasma Fractionating Centres allocate
resources for improving the quality of plasma as a raw
material to linked BCSU in the form of manpower,
equipment, logistics, etc.
5.4 To encourage training programs to ensure proficiency,
accreditation and other changing quality parameters
from time to time.
5.5 To encourage higher standards and uniformity,
External Quality Assessment Scheme (EQAS) shall be
introduced, through the referral laboratories approved
by the National Blood Transfusion Council.
5.6 To ensure complete process control with sound
documentation system, to inculcate data sharing and
create opportunities to promote learning and growth.
5.7 To collate and analyze the data and share with all stake-
holders, regularly as a part of the larger quality manage-
ment initiative in the area of plasma fractionation.
1. National Policy for Access to Plasma Derived Medicinal
Products from human plasma for clinical therapeutic use:
Adden dum to National Blood Policy 2003. Department of AIDS
Control, Ministry of Health and Family Welfare, Government of
India; Published in June 2014, NACO, 2014.
Ethical Issues in Transfusion
Medicine
Kusum K Thakur, Achhar Singh
Introduction
Blood transfusion is an altruistic medical act of ethical
responsibility.1 It involves a moral responsibility towards
both donors and patients. Decisions must be based on
four principles: respect for individuals and their worth,
protection of individuals’ rights and well-being, avoidance
of exploitation, and the Hippocratic principle of premium
non nocere or “first do no harm”. The practice of transfusion
medicine involves a number of ethical issues because blood
comes from human beings and is a precious resource with
limited shelf life. The framers and the users of guidelines
must be aware of the potential ethical conflicts inherent in
many medical decisions, and the guidelines must reflect a
thoughtful consideration and balancing of issues.2
Ethics is a dynamic process in relation to the state of
scientific knowledge, public awareness and the local laws,
at any given time and place. This is clear when we review
the history of transfusion ethics.3 Since the second half of
the 1980s, transfusion medicine has gradually taken on
an increasing autonomous place in the world of medical
specialties, continuing its evolution from its initial, mainly
immunohematological role, towards new diagnostic, clinical-
therapeutic and research activities. Currently, in addition to
direct care activities for patients and support of specific highly
specialized care processes, transfusion medicine ranges from
the collection and banking of hematopoietic stem cells to the
banking of tissues and cell therapy.4
Transfusion safety goes beyond the intrinsic safety of the
transfused therapeutic products, depending on a series of
closely interconnected processes which start with the donor
and finish with the recipient. The systematic recording of
transfusion outcomes and adverse events associated with
transfusion therapy constitutes the afferent branch of every
hemovigilance system and plays a fundamental role in the
search for and the identification of possible strategies for
improvement. In contrast, the purpose of the efferent branch
of these systems is to achieve a constant, global improvement
in the transfusion process through an analysis of the data
74
collected and their possible changes over time, to identify
possible corrective or preventive strategies based on objective
evidence. Therefore, it seems clear that the multi-faceted
and complex transfusion process, with its unique global
organizational and management framework, is not possible
without a systematic approach aimed at raising quality and,
therefore, at continuous improvement.4
The analysis of the available literature on ethical issues in
transfusion medicine is divided into the following points:4
• Clinical appropriateness
• Information and consent to/refusal of transfusion
• The development of institutional programmes of blood
management
• Confrontation between individual’s rights and public safety
• Financial compensation for blood donations
• The safety of underage donors
• The collection and storage of blood products: Biobanks.
International Society of Blood
Transfusion code of ethics
In 1980 the International Society of Blood Transfusion (ISBT)
endorsed its first formal code of ethics. It was later also
endorsed and adopted by the World Health Organisation and
the League of Red Crescent Societies. A revised code of ethics
for blood donation and transfusion was endorsed in 2000,
with inputs from various concerned organizations. It gave
recommendations regarding the ethical responsibilities of
the donor, the collection agency and the prescribing authority
toward the well-being of the recipient and the community at
large.5 This code is reproduced below:
A code of ethics for blood donation
and transfusion
The objective of this code is to define the ethical principles
and rules to be observed in the field of transfusion medicine.
282  Section 4  Miscellaneous
• Blood donation, including hematopoietic tissues for
transplantation shall, in all circumstances, be voluntary
and nonremunerated; no coercion should be brought to
bear upon the donor. The donor should provide informed
consent to the donation of blood or blood components
and to the subsequent (legitimate) use of the blood by the
transfusion service.
• Patients should be informed of the known risks and
benefits of blood transfusion and/or alternative therapies
and have the right to accept or refuse the procedure. Any
valid advance directive should be respected.
• In the event that the patient is unable to give prior informed
consent, the basis for treatment by transfusion must be in
the best interests of the patient.
• A profit motive should not be the basis for the establishment
and running of a blood service.
• The donor should be advised of the risks connected with
the procedure; the donor’s health and safety must be
protected. Any procedures relating to the administration to
a donor of any substance for increasing the concentration
of specific blood components should be in compliance
with internationally accepted standards.
• Anonymity between donor and recipient must be ensured
except in special situations and the confidentiality of
donor information assured.
• The donor should understand the risks to others of donating
infected blood and his or her ethical responsibility to the
recipient.
• Blood donation must be based on regularly reviewed
medical selection criteria and not entail discrimination of
any kind, including gender, race, nationality or religion.
Neither donor nor potential recipient has the right to
require that any such discrimination be practised.
• Blood must be collected under the overall responsibility of
a suitably qualified, registered medical practitioner.
• All matters related to whole blood donation and
hemapheresis should be in compliance with appropriately
defined and internationally accepted standards.
• Donors and recipients should be informed if they have
been harmed.
• Transfusion therapy must be given under the overall
responsibility of a registered medical practitioner.
• Genuine clinical need should be the only basis for
transfusion therapy.
• There should be no financial incentive to prescribe a blood
transfusion.
• Blood is a public resource and access should not be
restricted.
• As far as possible the patient should receive only
those particular components (cells, plasma, or plasma
derivatives) that are clinically appropriate and afford
optimal safety.
• Wastage should be avoided in order to safeguard the
interests of all potential recipients and the donor.
• Blood transfusion practices established by national or
international health bodies and other agencies competent
and authorized to do so should be in compliance with this
code of ethic.
Some important points are being given in details as follows:
ETHICAL ISSUES RELATED TO DONORS
The National Blood Policy in India relies heavily on voluntary
blood donors, as they are usually assumed to be associated
with low levels of transfusion‐transmitted infections (TTIs).
In India, it is mandatory to test every unit of blood collected
for hepatitis B, hepatitis C, HIV/AIDS, syphilis and malaria.
Donors come to the blood bank with altruistic intentions. If
donors test positive to any of the five infections, their blood is
discarded. Although the blood policy advocates disclosure of
TTI status, donors are not, in practice, informed about their
results. The onus is on the donor to contact the blood bank.
Out of approximately 16000 donations in the past 2 years,
438 tested positive for TTI, including 107 for HIV. Only 20%
of the donors contacted the blood bank; none of them were
HIV positive. Disclosure by blood banks of TTI status by
telephone or mail has resulted in serious consequences for
some donors. Health providers face an ethical dilemma, in
the absence of proper mechanisms in place for disclosure
of test results, regarding notification to donors who may
test positive but remain ignorant of their TTI status. Given
the high cost of neglecting to notify infected donors, the
authors strongly recommend the use of rapid tests before
collecting blood, instead of the current practice, which takes
3 hours to obtain results, and disclosure of results directly to
the donor by a counsellor, to avoid dropouts and to ensure
confidentiality.6
• Blood donation as a gift: The WHO recommends that
national blood services should be based on voluntary, non-
remunerated blood donation. No one should be forced to
donate, for family or economic or any other reason. The
trade of human blood and body parts is unethical. “The
dignity and worth of the human being should be respected.” 
• Nonremunerated blood donation is considered as a gift
and the blood center has a right to accept or defer it if
unacceptable.
• Donor deferral: Might appear as discrimination and a
violation of a human right, but the patient’s right to safer
blood is more important than the donor’s right to not to
discriminated against, as blood centers are made to help
patients and not donors.
• Donor confidentiality, donor notification and donor
consent: Donor confidentiality is an important issue.
Personal information disclosed by the blood donor during
the course of a pre-donation interview and information
obtained from the various tests performed on the donated
component, are expected to be held in confidence by the
donor center.

• Donor screening and testing used to be simple: Today’s
donors are asked intimate questions about their lifestyles
and put through a battery of laboratory tests. This has had
significant repercussions for the relationships between
blood centers, blood donors, physicians and patients.
The blood donor, an ostensibly healthy individual until
notified of an abnormal result by the blood center, may
seek a physician’s advice and doubt the creditability of the
testing procedure and deferral policies. A more specific
test might turn out to be negative and the donor may be
labeled as healthy. This donor might return to the blood
center asking for compensation for the unnecessary
mental anguish and the expenses incurred and might
never donate again.
• Blood safety depends partly on the information provided
by the donor and it is also the donor’s ethical duty to
provide truthful information. It is unethical to wilfully
conceal information about high-risk behavior or medical
history.2
ETHICAL ISSUES RELATED TO PATIENTS
Ethical issues related to patients include access to risk-free
safe blood free of charge or need of replacement, informed
consent for transfusion, the right to refuse the transfusion,
and the right to be informed if harmed.2
• Consent for transfusion: Consent for transfusion has to be
informed consent. The patient should be informed of the
known risks and benefits of transfusion, and alternative
therapies such as autologous transfusion or erythropoietin.
Only then should the consent be documented. If the
patient is unable to give prior informed consent, the basis
of treatment by transfusion should be in the best interests
of the patient.
• Right to refusal: The patient’s right to refuse blood
transfusion should be respected. Some religious sects such
as Jehovah’s Witnesses do not accept blood transfusions.
Followers of this belief live in India as well and there have
been instances of blood refusal here.
• Right to be informed if harmed: If the patient has been
transfused blood and components that were not intended
for him/her, whether harmed or not, he/she has the right
to be informed. Similarly a patient who has inadvertently
received blood positive for a transfusion transmissible
marker has a right to be informed and given due
compensation.
ETHICAL ISSUES RELATED TO BLOOD
ESTABLISHMENTS
A profit motive should not be the basis of establishing and
running blood transfusion services. Wastage should be
avoided to safeguard the interests of all potential donors and
recipients.
Chapter 74  Ethical Issues in Transfusion Medicine  283
Indian Scenario of Ethical Issues in
Transfusion Medicine
With the rising awareness of ethical issues in every field of
medical care and research in India, awareness is growing in
the field of transfusion medicine as well. But we are nowhere
near the international code of ethics.
In the 1990s, in response to public interest litigation a
Supreme Court order banned professional blood sellers and
directed the government to formulate a national blood policy.
The National Blood Transfusion Council, with the National
Blood Policy as a tool, and the Drugs Controller, with the help
of the Drugs and Cosmetics Act, now aim to ensure blood
safety and ethical transfusion practices in India.
Currently under the Drugs and Cosmetics Act it is manda­
tory to test blood for anti-HIV 1 and 2, anti-HCV, HBsAg, RPR
for syphilis and malaria.7 Consent for testing is taken and
the donor is given the option of receiving the results—this is
mandatory in some countries such as the US and UK.
Until recently donors were not informed because specific
consent for testing was not taken,8 and the screening tests had
relatively high false positive rates, which could cause panic.
No confirmatory tests were required. So the donation system
was projected as anonymous and unlinked and adequate
counseling was not available. The National Blood Policy of
2002 has addressed this gap.9
The Code of Medical Ethics that is binding on doctors,
honors confidentiality. However, in a court of law in India,
this privilege is not absolute but qualified. Doctors can reveal
information in the interest of individual or general welfare of
society and when there is no mal-intention.
Ethical issues are mostly violated in relation to the patient
in India. Patients all over the country do not have access to
safe blood, free of charge, or the option of giving consent and
choosing safer alternatives. With the National Blood Policy,
a decision was taken to improve transfusion services all over
the country and create greater awareness about transfusion
issues. The policy must also address all the other issues
in the international code of ethics for blood donation and
transfusion to make India achieve international standards.
National AIDS Control Organisation (NACO) is working
hard to improve blood transfusion services (BTS) in India.1
Now counsellors have been provided for donor counseling
and notification in almost all blood banks. But various studies
in donor management have shown that there is still more to be
done to attract donors to collect their test reports and do the
follow-up as suggested by counselor.
Recently hemovigilance programme has been launched
in India by government to monitor blood safety and CMEs
are being conducted in various medical institutes to apprise
clinicians about ethical issues related to patients.
FUTURE PROSPECTIVES
As per Roman literature, there are at least two other ethically
important issues to be considered in transfusion medicine,
284  Section 4  Miscellaneous
given its organizational-procedural complexity. It is
precisely this complexity which requires refined instruments
of analysis also from the ethical point of view. We are
referring to the methodology known as health technology
assessment (HTA) and to projects of organizational ethics in
logic of governance of transfusion medicine. HTA is an
area of multidisciplinary research which aims to provide a
valid response to the complexity of health care, through a
“structured” or “multidisciplinary” process of analysis and
decisions: it is “structured”, because the process involves
the collection and analysis of data to support decisions
through a systematic review of the scientific literature; it is
“multidimensional”, in that the impact of the technology
must be evaluated at various levels: clinical, financial,
organizational, social, psychological, legal and ethical.
Organizational ethics, on the other hand, is configured
as “the process that tackles ethical problems related to the
care of patients, to the business, financial and managerial
sectors of health care organizations as well as to professional,
educational and contractual dynamics which influence the
functioning of the organisations”. HTA and organizational
ethics could have useful roles in the optimization of
organizational patterns and choices which a complex reality
such as transfusion medicine requires, taking into account
the substantial lack of contributions on this specific issue.
CONCLUSION
Margaret Somerville who, in an article written a few years
ago, stated: “an essential characteristic of all health care
services, including transfusion services, is that ethics are
included... ethics should be intrinsic to the governance and
management of the system, that is, to all the decisions, actions
and processes implemented within it.10
REFERENCES
1. Pal R, et al. The quest for an Indian blood law as of blood
transfusion services regulatory framework. Asian Journal of
Transfusion Science Year. 2011;5(2):171-4.
2. Elhence P. Ethical issues in transfusion medicine. Indian J Med
Ethics. 2006;3:87-9.
3. Rossi EC, Simon TL. Transfusion in the new millennium.
In: Simon TL, Dzik WH, Snyder EL, Stowell CP, Strauss RG,
(Eds).  Rossi’s principles of transfusion medicine.  3rd edn.
Philadelphia: Lippinkott William and Wilkins; 2002. pp.1-13.
4. S Dario, et al. Ethical and deontological issues in Transfusion
Medicine. Blood Transfusion. 2013;11(1):14-25.
5. International Society of Blood Transfusion [homepage on
the Internet].  A code of ethics for blood donation and blood
transfusion. [cited 2006 June 15]. Available from: http://www.
isbt-web.org/files/documentation/code_of_ethics.pdf
6. Choudhary LP, Tetali S. Ethical challenges in voluntary blood
donation in Kerala. Indian J Med Ethics. 2007;33:140–2.
7. Government of India. Drugs and Cosmetics Rules, 1945
(amended till 30th June 2005). [cited 2006 July 20]. Available
from: http://www.cdsco.nic.in/html/Drugs & CosmeticAct.pdf
8. Watwe JM. Disclosure of confidential medical information.
Issues Med Ethics. 1998;6:56-7.
9. Ministry of Health and Family Welfare. National Blood
Policy. National AIDS Control Organization, Government of
India 2002. [cited 2006 July 20]. Available from: http://www.
nacoonline.org/prog_policyblood.htm
10. Somerville MA. Ethical issues and challenges in implementing
a new blood system. Transfus Med Rev.1998;12:162-74.
Gel Based Technology: Past,
Present and Future

Nidhi Bhatnagar
75
Transfusion services in the India are currently undergoing
dynamic changes in an effort to optimize patient blood
management and ensure rapid availability of the safest, most
compatible blood products possible. Providing precisely
matched blood to transfusion patients to reduce the risk of
adverse events and improve transfusion outcomes remains
the ultimate goal of immunohematology practice. This is
particularly important when managing patients who receive
chronic transfusions, including those with sickle cell disease,
thalassemia, or chronic anemia, who are more likely to
produce alloantibodies.
Historically, red blood cell agglutination testing has been
conducted using conventional tube techniques due in large
part to their simplicity. Although suitable for individual testing, Figure 1:  Principle of the gel test column
the tube method presents some limitations, including low agglutination technology
reproducibility, the potential for human error, and inefficient
personnel management. For example, tube shaking methods • Large agglutinates remain on or near the top of the gel
may differ by user and affect the grading and interpretation interface. Smaller agglutinates pass partway through the
of the results. Other challenges include repeated washing gel, depending on size.
steps, resulting in loss of red blood cells (RBCs) and weakly • Unagglutinated cells pass to the base of the microtube.
bound antibody; difficulty detecting mixed-field populations; Column agglutination technique (CAT), methods have
and providing accurate RBC typing in recently transfused emerged in the last two decades, and are now replacing
individuals. Additionally, the limited capacity for automation manual tube methods due in large part to their flexibility,
inherent with tube testing results in increased frequency of efficiency, and reliability. The original technique was based
transcription errors.1 Tube testing also is considered fairly on the principle of gel filtration for separation of red blood
labor-intensive, resulting in high costs and inadequacy for cells from human blood.3 It was found that the principle of
large-scale implementation.2 gel centrifugation could be modified for use as a serological
tool using Sephadex G100 superfine or Sephadex G200
Principle of Gel Technology (Fig. 1) Superfine. Originally described by Yves Lapierre et al.4 in
1985, the aim of this technology is to standardize red blood
• The serum and cell reaction takes place in a microtube.
cell agglutination reactions, and by trapping the agglutinates,
• This microtube consists of a reaction chamber that narrows
to permit simple and reliable reading. The column consists of
to become a column.
special microtubes containing a dextran gel matrix. Red blood
• Each column contains sephadex gel suspended in a buffer
cells and serum or red blood cells alone are dispensed into
solution.
the microtubes, incubated if necessary, and then centrifuged
• Depending on the configuration of the card, the gel is
under strictly controlled parameters. The gel within the
premixed with antisera/AHG/other reagents.
microtubes acts as a sieve, unagglutinated red blood cells
• The sephadex gel matrix acts as a sieve.
form a pellet at the bottom of the microtube, and agglutinated
286  Section 4  Miscellaneous
red blood cells are trapped in the gel. The gel may be neutral or
contain specific reagents such as AHG or specific antibodies
(anti-A, -B, anti-D, anti-Kell, etc.). Reactions are easily visible
and may be graded (Fig. 2).
Practical applications of
gel technology
When performing the antiglobulin tests, no washing of the
red blood cells is required because during centrifugation, the
cells are separated from their suspension medium and serum
as they pass into the microtube. Red cells sensitized by IgG
or complement components react with the AHG contained in
the gel and the resulting agglutinates are retained within the
matrix. The serum fraction does not, therefore, make contact
with the AHG impregnated gel, so that neutralization of the
AHG reagent is avoided.
This technique also obviates the need to use pre-sensitized
control cells to check negative reactions. Neutral cards can
be used as part of antibody screening for a two stage enzyme
treated cell technique.
Reactions using gel techniques are stable for at least
48 hours and have the added facility of being able to be
photocopied, thereby providing a permanent record for
future reference. This system is currently marketed as the ID-
Microtyping System (DiaMed-GB) in 1988.
Another system is also available based on column
technology-the Ortho Biovue System (Ortho Diagnostic). This
column contains a density gradient comprising a combination
of a macromolecular density barrier and glass microspheres.
The system is currently available as microtubes with columns
containing AHG or neutral density gradients. Again, this
system offers the ability to perform a “no wash” antiglobulin
test.
It has been shown that the sensitivity of Dia Med ID gel
system is superior to that of conventional tube tests without
any loss of specificity.4-6 A comparison of different techniques
for routine antibody screening and identification showed
increased antibody detection rate and decrease in the number
Figure 2:   Grading of agglutination
of nonspecific antibodies and false positive reactions. These
techniques included an automated polybrene technique,
two-stage papain technique, microtiter plate IAT, and a spin
tube low ionic strength IAT. Antibody titers showed increased
reaction strength and so titer scores were higher than tube IAT
titers, suggesting an increased sensitivity for the gel system.
Use in a routine hospital blood bank laboratory for red cell
phenotyping (ABO, Rh, Kell, M and N), direct antiglobulin
testing (DAT), antibody screening and indirect antiglobulin
test compatibility testing showed that care must be taken to
ensure that no greater than a 1% suspension of red blood cells
is used and that problems may be encountered in patients
who are DAT positive.
It has also been shown, however, that serological
assessment of drug induced immune hemolytic anemia is
considerably improved by using the gel system.7
Gel column agglutination, which has gained popularity
in recent years, also has the unique advantage of detecting
mixed-field agglutination patterns, because the high-density
media allows a clear separation between agglutinated and
nonagglutinated cells, thus indicating the presence of a dual
population of red blood cells.8 It was shown that gel column
agglutination is superior to other commonly used serology
methods, including solid phase red cell adherence assay
(SPRCA) in the detection and monitoring of mixed-field
reaction patterns. 
Also, in certain situations with antibody and/or
complement attached to RBCs, the gel column method
appears to be more accurate in estimating the degree of RBC
sensitization in direct antiglobulin testing.9  On a practical
level, the stability of the gel-based agglutination method’s
endpoint is another important advantage of the test. The
results can be read and reviewed at a later time, whereas the
endpoint of the manual tube method is stable for less than
an hour. This stability allows the less-experienced technician
to review ambiguous results with a supervisor and enables
documentation in the patient’s chart with photographs of the
gel card.
Advantages of Gel Technology
• No cell washing is involved
• Each Gel microtube is already being filled up with AHG
reagent with air-tight seal→ so no chance of contamination
of AHG reagent →No erroneous result.
• 25 minute Test, without Hazardous cell washing step-
Labor and Time saving.
• No requirement of sensitized check cells—Time and cost
saving.
• Result remains as it is for at least 48 hours in RT.
• No degradation, easy to supervise. Gel cards can be
scanned or photocopied for permanent documentation.
• Helps in accreditation process.
• Further option for automation is available.
• It can help in medicolegal cases.
 Chapter 75  Gel Based Technology: Past, Present and Future  287
Past, Present and Future
In the past when the technology was newly launched in
market, it was used for performing basically Indirect Coombs
test. The blood bank technicians found this technique very
convenient, easy to perform, standard, reproducible, and
useful from documentation point of view.
At present, this technology is being used globally and well
accepted among all the transfusion medicine fraternity. The
technology is being currently used in almost 30% of the blood
banks across India. Majority of the blood banks are performing
Coombs cross match, DCT, ICT, etc. Some of the Blood
banks are using this technology not only for crossmatch but
also for extensive tests like—Antibody screening, Antibody
identification, Weak D testing, etc. The technology is being
used for blood grouping also as there are different formats of
cards available now like forward grouping only, forward and
reverse grouping, reverse grouping only, newborn grouping,
cards with two clones of Anti-D, A1 and H Lectin cards.
At present, the antibody screening test is not a routinely
performed in all the blood banks, but, with the help of Gel
technology this can be performed very easily and blood banks
arrange the requisite type of blood to patient easily in case of
positive antibody screening with the help of own inventory or
referring to the other blood banks in near vicinity which are
performing the antibody identification and antigen typing of
the donors.
Presently, automation is also available for this technology,
but it is being used for very few parameters. The future of Gel
technology is the automation of all the immunohematology
tests. With automation, we can perform the antigen typing
of donors and can have a central donor data base, so that
in case of emergency, desired donors can be called and safe
transfusion can save the life of patient.
Thus, column agglutination technology offers several
technical benefits including pre-dispensed reagents,
omission of the washing step in the antiglobulin phase,
and stability of the reaction in the column.  Although there
are differences in sensitivity and specificity between these
automated methods, all commercially available gel column
agglutination systems have proven to be highly accurate with
a sensitivity >97.58% and a specificity >99.93%.10,11
It provides a new approach which may, by its sheer simplicity
of use and standardization of technique, facilitate the working
practices of transfusion laboratories and thus enhance the
quality of the service provided to both patients and clinicians.
References
1. Mistry H. Automation in UK blood transfusion laboratories:
Strategies for error reduction.  Perspectives in Transfus Med.
2013;3:7-10.
2. Rumsey DH, Ciesielski DJ. New protocols in serologic testing:
a review of techniques to meet today’s challenges.  Immuno­
hematology. 2000;16(4):131-7.
3. Kanura T, Kurashina S, Nakao M. A gel filtration technique for
separation of erythrocytes from Lab Clin Med. 1974;83:840-4.
4. Lapierre Y, Rigal D, Adam J, et al. The gel test: A new way to detect
antigen-antibody reactions. Transfusion. 1990;30:109-13.
5. Hitzler W, Shomig-Breckner H, Mathias D. Gel centrifugation
test-a new micro method for blood group typing and antibody
screening. Arzthide Laboratorium. 1989;35:89-92.
6. Bromilow IM, Adams KE, Hope J, Eggington JA, Duguid JKlM.
Evaluation of the ID-gel gest for antibody screening and
identification. Transfusion Med. 1991;1:159-61.
7. Salama A, Berghofer H, Mueller-Eckhardt C. Detection of cell-
drug (hapten)-antibody complexes by the gel test. Transfusion.
1992;32:554-6.
8. Summers T Jr, Johnson VV, Stephan JP, Johnson GJ, Leonard
G.The value of automated gel column agglutination technology
in the identification of true inherited D blood types in massively
transfused patients. Transfusion. 2009;49(8):1672-77.
9. Dittmar K, Procter JL, Cipolone K, Njoroge JM, Miller J, Stroncek
DF. Comparison of DATs using traditional tube agglutination
to gel column and affinity column procedures.  Transfusion.
2001;41(10):1258-62.
10. Cid J, Nogués N, Montero R, Hurtado M, Briega A, Parra R.
Comparison of three microtube column agglutination systems
for antibody screening: DG Gel, DiaMed-ID and Ortho
BioVue. Transfusion Med. 2006;16(2):131-6.
11. Chang C, Brown M, Davies L, Pointon L, Brown R, Barke D.
Evaluation of Erytra fully automated analyser for routine use in
transfusion laboratory. Transfus Med. 2014;24(1):33-8.
Automated Blood Grouping
and Antibody Screening
Ashish Jain
In 1901, Karl Landsteiner’s elucidation of the ABO blood
group system established the basis for numberless discoveries
of new human blood group antigens and antibodies during
the past hundred years. The discovery and identification of
human blood groups represented a milestone for a successful
administration of compatible blood transfusions and
therefore founded the introduction of immunohematology
as new scientific subject.1 Immunohematology covers the
analysis of blood group antigens and antibodies and their
interactions in health and disease.2 For many years, the
manually performed tube tests were the only standard
in immunohematologic laboratories for all routine and
scientific questions. These tube tests were later followed
and supplemented by more sensitive and specific manual
methods, such as solid phase or gel centrifugation techniques.
Only since the past few years, partly or fully automated systems
for blood group typing and cross-matching were introduced
in immunohematology to a greater extent.1 Now-a-days,
an increasing amount of immunohematologic laboratories
uses automated systems for blood group typing and cross-
matching in addition to the manual work strategies.
Automation in serologic testing
Automation of serologic testing has many advantages,
including improved quality management. With fewer manual
steps, standardization of testing, and the ability to review
saved images of test results, errors are reduced and problems
are more easily traced. Purchasing, installing, validating, and
using automation present challenges to transfusion service
personnel more familiar with manual testing.3
Factors to be considered in equipment selection should
include: 3
• Platform and test methods
• Physical space and electrical requirements
• Tests to be automated
• Stat processing capability
• Backup testing method and costs
76
• Routine turnaround time
• Quality control requirements
• Cost for operation
• Ease of operation
• Laboratory information system interface capability and
cost
• Training requirements and costs
• Vendor assistance in validation and training
• Maintenance and service agreements
• Mean time between failures.
Chizhevsky et al.4 demonstrated that using an automated
system for ABO/Rh(D) typing and unexpected antibody
screening improves productivity compared to using
the manual methods by decreasing the hands-on time
required. Sarkozi et al.5 reported that the introduction of a
robotic system for peri-analytical automation substantially
improved productivity and decreased operational costs in
a clinical chemistry laboratory. This system enabled them
to significantly increase their workload while reducing
personnel. A few of the automated platforms available today
are described in Table 1.3
Shin et al.6 compared automated and manual results where
they found that the concordance rates were 100%, 99.6%, and
100% with respect to forward ABO typing, reverse typing, and
Rh typing tests, respectively. Lee et al.7 reported concordance
rates between AutoVue Innova and manual methods of 99.6%
and 100% for ABO/ Rh(D) typing and unexpected antibody
screening, respectively. Kim et al.8 analyzed 136 samples
using the unexpected antibody screening test and found
that 2 results were positive only on the AutoVue Innova and
not the LISS/Coombs card. Han et al.9 reported the use of
automated equipment dramatically reduced the risk priority
number compared to manual processes.
Solid-phase immunoassays
Solid-phase immunoassays are test systems in which one
of the reactants (either antigen or antibody) is immobilized
 Chapter 76  Automated Blood Grouping and Antibody Screening  289

to a solid support before test initiation.10 During testing, the
immobilized component captures additional reactants in the
liquid phase and binds them to the solid phase. Solid-phase
red cell adherence (SPRCA) assays have evolved from several
techniques as an alternative to conventional hemagglutination
assays. Rosenfield et al.11 were among the first investigators to
describe SPRCA techniques for use specifically in the blood
bank laboratory. The use of stable pre-dispensed reagents
also permits SPRCA assays to evolve into fully automated test
procedures.10 Reagent red cell membranes that have been
immobilized to the surfaces of polystyrene microtitration
plates are used to capture erythrocyte-specific IgG antibodies
from patient or donor sera or plasma. After a brief incubation
period, unbound components are washed from the wells
and anti-IgG-sensitized indicator red cells are added. In a
positive reaction, centrifugation causes the indicator red cells
to adhere immunologically to the captured red cell-specific
antibodies (via antibody bridging), resulting in the total or
partial adherence of the indicator red cells over the surfaces
of the wells. In the absence of detectable antigen-antibody
interactions (negative test), the indicator red cells are not
impeded during centrifugation and pellet to the bottom of
the wells as tightly agglutinated cell buttons. A positive test
shows adherence of indicator red cells over part, or all, of the
reaction surface. A negative test shows a button of indicator
red cells at the bottom of the test well.
Erythrocyte-magnetized technology
The erythrocyte-magnetized technology (EMT; Diagast, Loos,
France) is an original and innovative technology based on the
magnetization of red blood cells (RBCs).12 The use of magnetic
beads to allow quick transfer of antigens or antibodies with
simple magnets has been an innovation force for automated
diagnostic devices in fields other than immunohematology.
This principle allows a highly reliable automation without the
need for centrifugation.13 The heart of EMT is the adsorption
of paramagnetic (magnetism occurring only in the presence
of an externally applied magnetic field) compounds on the
membrane of the RBCs. Thus, when placed on a magnetic
plate after contact with antibodies, reactive and nonreactive
magnetized RBCs are rapidly pulled to the bottom of the well.
A final phase of shaking reveals positive or negative reactions
(Fig. 1).

Table 1: Features of fully automated immunohematology analyzers3

Galileo ECHO ProVue Tango
Test menu
ABO Yes Yes Yes Yes
D Yes Yes Yes Yes
Antibody screen Pooled, 2-cell, 3-cell, 4-cell 3-cell plus control Pooled, 2-cell or 3-cell Pooled, 2-cell or 3-cell
Antigen typing Rh phenotype test of Rh/Kell phenotype Yes Yes
record/antigen screening
Panels Yes, 3 preoated panels Yes, precoated panels Yes Yes
available
Other testing Weak D, DAT, CMV, Weak D, DAT Weak D, DAT Weak D, DAT
syphilis
Cross-match IgG IgG IgG IgG
Unit ABO/D confirmation Yes Yes Yes Yes
System liquid
PBS PBS Gel card diluent
Can be filled while Can be filled while
instrument is processing instrument is processing
Can accommodate a 20 L
saline cube
Turn around time
Time for first ABO/D 22 minutes 11 minutes 35-40 minutes 17 minutes
Time for antibody screen 40 minutes 20 minutes 35-40 minutes 31 minutes
Specimens/hour Variable, dependent on Variable, dependent on Variable, dependent on 12 type and screens/64
number of samples and number of samples and number of samples and minutes
test mix; average of 60 test mix; average of 16 test mix
type and screens/hour type and screens/hour
290  Section 4  Miscellaneous

Figure 1  Blood grouping using EM technology in automated equipment

 Chapter 76  Automated Blood Grouping and Antibody Screening  291
Validation of automated systems3
Vendors provide installation instructions and recommenda­
tions for verification of instrument operations. Testing should
include a sufficient number of positive and negative antibody
screens; various ABO and D types, including specimens with
dual populations; and a comparison of results with those
obtained using the current method. The validation should
also include a provision for how discrepant results will be
resolved. The manufacturer’s instructions for quality control
and periodic maintenance should be followed. Reviewing
protocols from other instrument users is helpful.
edge of automation
In a study by South et al.14 to compare error potentials
of commonly used manual (e.g. tiles and tubes) versus
automated (e.g. ID-GelStation and AutoVue Innova) Group
and Screen (G and S) methods, routine group and screen
(G and S) processes in seven transfusion service laboratories
(four with manual and three with automated G and S
methods) were analyzed. Their study provided quantitative
evidence on how automation could transform pretransfusion
testing processes by dramatically reducing error potentials
and thus would improve the safety of blood transfusion.
References
1. Cassens U. Automation in transfusion medicine. Transfus Med
Hemother. 2007;34:313-4.
2. Cruse JM, Lewis RE. Immunohematology; in: Historical Atlas
of Immunology. London, Taylor and Francis. 2005. pp. 207-23.
3. Butch SH. Automation in transfusion service. Immuno­
hematology. 2008;24:86-92.
4. Chizhevsky V, Esagui D, Delaflor-Weiss E. Evaluation of an
automated system for ABO/D typing and RBC antibody
detection system in a hospital transfusion service. LabMedicine.
2005;36:29-31.
5. Sarkozi L, Simson E, Ramanathan L. The effects of total
laboratory automation on the management of a clinical
chemistry laboratory. Retrospective analysis of 36 years. Clin
Chim Acta. 2003;329:89-94.
6. Shin SY, Kwon KC, Koo SH, Park JW, Ko CS, Song JH, et al.
Evaluation of two automated instruments for pre-transfusion
testing: AutoVue Innova and Techno TwinStation. Korean J Lab
Med. 2008;28:214-20.
7. Lee SH, Jeong J, Jeong US, Kim MS, Jeong YJ, Wee JH,
et al. Experience with the automatic blood bank instrument
AutoVue Innova. Korean J Blood Transfus. 2008;19:43-8.
8. Kim SH, Nam DH, Yang JH, Yoon SY, Kim YK, Lee KN, et al.
Evaluation of the automatic blood bank instrument AutoVue
Innova for antibody screening. Korean J Blood Transfus.
2008;19:140-5.
9. Han TH, Kim MJ, Kim S, Kim HO, Lee MA, Choi JS, et al. The role
of failure modes and effects analysis in showing the benefits of
automation in the blood bank. Transfusion. 2013;53:1077-82.
10. Growe GH, Galenza J, Mah H, Whitehead R, Godolphin W.
The implementation and use of automated group and screen
procedures in a hospital transfusion laboratory. Transfus Med
Rev. 1996;10:144-51.
11. Rosenfield RE, Kochwa S, Kaczera Z. Solid phase serology for
the study of human erythrocytic antigen-antibody reactions.
Proceedings of the 12th Congress of the International Society
of Haematology and the 15th Congress of the International
Society of Blood Transfusion, Paris, France, 1978. pp. 27-33.
12. Bouix O, Ferrera V, Delamaire M, Redersdorff JC, Roubinet
F. Erythrocyte-magnetized technology: an original and
innovative method for blood group serology. Transfusion.
2008;48:1878-85.
13. Shin KH, Kim HH, Chang CL, Lee EY. Economic and workflow
analysis of a blood bank automated system. Ann Lab Med.
2013;33:268-73.
14. South SF, Casina TS, Li L. Exponential error reduction in pre­
transfusion testing with automation. Transfusion. 2012;52:
81S-7S.
Molecular Genotyping and
its Applications to
Transfusion Medicine
Swati Kulkarni
Antigen antibody based hemagglutination technique
has been the gold standard method to detect presence or
absence of blood group antigens for over a century now.
These techniques are simple, cheap, familiar and highly
effective means of defining blood group status. It requires
simple equipment. However, hemagglutination has certain
limitations. It is a subjective test which requires reliable
antisera. It is labor intensive and time consuming. Except for
routine ABO and RhD grouping, potent antisera for extended
phenotyping are very costly, weakly reacting and unavailable
for some blood group system like Dombrock, Coltan, etc.
For some blood group antigens, the antisera are poorly
standardized polyclonal human serum which is available in
limited stocks. Hemagglutination technique has restricted
ability to predict RhD zygosity, define weaker variants of ABO
and Rh, RBC phenotyping in multi-transfused patients and
imunoglobulin coated RBCs.
Till date, 339 antigens have been discovered on human
red cells of which 297 are clustered within 33 blood groups
systems and the remaining are accommodated in the
Collections, 700 Series of Low Incidence Antigens or the
901 Series of High Incidence Antigens.1 Blood groups are
inherited, polymorphic, structural characteristics located on
proteins, glycoproteins or glycolipids on the surface of RBC
membrane. RBCs carrying a particular antigen, can elicit an
immune response that causes problems in clinical practice
such as patient/donor blood transfusion incompatibility,
maternal fetal incompatibility and autoimmune hemolytic
anemia. The knowledge of molecular bases associated with
blood group antigens and phenotypes enables us to consider
prediction of presence or absence of blood group antigens,
thereby overcoming limitations of hemagglutination.
Numerous molecular events generate blood group antigens
and phenotypes, however, most antigens result from a single
nucleotide change, and simple DNA based assays can be used
to predict the presence or absence of blood group antigen.
High-through put platforms provide a means to test relatively
large number of donors, thereby increasing antigen negative
77
inventory which will result in giving antigen matched blood
to patients requiring multiple transfusions.
Some of the major applications of molecular genotyping in
immunohematology are:
Solving ABO blood group
discrepancies
Discrepancies in ABO blood groups among donors and
patients arise when cell grouping and serum grouping results
do not match. This may often be due to expression of variant
ABO allele. Such serological discrepancies can be resolved
by molecular genotyping of ABO gene thus confirming that
discrepancy is due to genetic variants.2 Such genotyping
methods can confirm subgroup status, help to elucidate new
variant alleles and understand the molecular mechanism
underlying the serological behavior of these genes. It is
also useful for distinguishing an acquired phenotype from
an inherited one. In case of Bombay and Para Bombay
phenotypes, inheritance pattern of ABO gene can only be
determined by molecular methods without performing time
consuming and laborious family studies.
Antigen Typing when Antibody-based
Typing Reagents are Unavailable or
Only Weakly Reactive
Agglutination tests depend upon the availability of specific
reliable antisera. Some of these blood group typing reagents
are not always readily available in large volumes for
serological typing of all blood group antigens or are weakly
reactive. For example, antisera to Fyb in Fyx, Kna, Knb are
often weakly reacting and testing becomes difficult. Also,
discrepancies in serological reactivity can occur between
different manufacturer’s reagents. Donors and patients
cannot be serologically tested for some blood group systems
like Colton, Dombrock, etc. as antisera are not available and
 Chapter 77  Molecular Genotyping and its Applications to Transfusion Medicine  293
therefore the identification of antigen negative unit becomes
difficult. Molecular methods help to define these antigens.
Identification of weaker and partial
variants of RhD gene
The Rh blood group discrepancies may arise when an
individual is a variant of D antigen. These variants are identified
as RhD negative or positive depending on the reagents in
use and techniques used in different laboratories. These
variants are of clinical importance and should be identified
as they may produce anti-D when transfused with normal D
positive red cells. Serological testing with two anti-D reagents
in blood banks may not identify D variants and also will not
distinguish between partial D from weak D.3 Identification
of these variants is important as these individuals are
considered as RhD positive as donor and RhD negative as
recipient of blood transfusion. In case of antenatal women, it
is essential to identify weak D/partial D status as the mother
is at the risk of developing anti-D followed by sensitization
to paternal antigens carried by the fetus. Molecular methods
are now developed to determine weaker and partial variants
of RhD. Our study in western Indian population has shown
that more percentage of RhD variants could be classified and
characterized using multiplex PCR compared to serological
methods using partial D kit (panel of six monoclonal anti-D).4
Many countries have developed molecular strategies to
identify common RhD variants present in their population.
Molecular assays will lead to identification of these variants
which will be of immense help in management of antenatal
women and donors.
RhD zygosity testing
RhD zygosity in RhD positive individuals is commonly inferred
from results of serological testing with anti-C, -c, -D, -E and -e
antisera. From the phenotype expressed and the frequency
of these antigens in population the most probable genotype
(MPG) is deduced and thus the D zygosity. This approach has
been a simple guess and discriminates indirectly between
RHD+/RHD+ and RHD+/RHD– and is therefore not very
accurate or reliable. The correct RhD zygosity can be predicted
by molecular studies. D zygosity determination by serological
and molecular testing has shown varying results.5 Kulkarni
et al. have reported 18% discrepancy in D zygosity between
serological MPG and hybrid box PCR assay among Indian
donors.6 The RhD zygosity determination is very important
in RhD negative pregnant women to predict the possible
RhD status of the fetus. If father is homozygous then all the
children will be RhD positive and if heterozygous, then there
is 50% chance of fetus being at risk. RhD negative pregnant
women can be reassured and managed less intensively if RhD
negative status of fetus was confirmed. Molecular analysis
will help in the identification of RHD zygosity of the father
by either determining the presence of Rhesus box using
sequence specific primers or by assessing the dosage of RHD
relative to a gene by real-time PCR.7, 8
Identification of DEL phenotypes
D-elute (DEL) phenotype has very low D antigenic sites which
may not be identified by IAT. These can be mistyped as RhD-
negative using routine serological testing. DEL phenotype
have been reported in literature to produce antibodies if
transfused to RhD negative recipient, hence their identification
is important. Absorption-elution or molecular techniques are
necessary for their identification. In Eastern Asia, between
10 and 33% of red cell samples found to be D negative by
conventional serological techniques are DEL variants.9
DEL phenotype which is associated with the presence of
mutated RHD gene can be molecularly screened. All donors
serologically typed as RhD-negative should be genotyped to
detect DEL variants.
Prenatal diagnosis of RhD in fetus
Monitoring RhD negative pregnant women at high risk of
HDFN relies on serial assessment of maternal IgG antibody
levels, Doppler ultrasound measurement and intrauterine
fetal sampling. RhD negative pregnant women with a
heterozygous partner can be managed differently depending
on the RhD status of the fetus.10
The conventional techniques used for prenatal testing
of fetal RhD status are mainly invasive such as chorionic
villus sampling, amniocentesis and cordocentesis. These
methodologies carry significant risk of complications
including pregnancy loss and increased maternal sensitiza­
tion.11
The discovery of cell free fetal DNA in maternal plasma
has opened up new possibilities for noninvasive prenatal
diagnosis. With the use of Real Time PCR methodology,
circulating fetal DNA has been detected robustly in plasma
of pregnant women, even in first trimester of pregnancy. Few
laboratories across the world now offer this test diagnostically
for management of alloimmunized pregnant women. Studies
are now being carried out to use this test diagnostically in
non alloimmunized pregnant women for restricting use of Rh
Immunoglobulin prophylaxis. Few studies on noninvasive
genotyping of fetal c, E and K have also been reported.12 Thus,
noninvasive fetal RhD typing can be used in management of
both Rh alloimmunized and nonimmunized pregnancies.
Typing of Blood Group Antigens in
Recently Transfused Patients
Agglutination tests prove unsuccessful in determining blood
group phenotypes in patients who have undergone a recent
blood transfusion due to the presence of donor RBCs in
patient circulation. DNA based typing of red cell antigens
overcomes this difficulty. DNA can easily be extracted from
294  Section 4  Miscellaneous
a post transfused patient as minor amounts of donor DNA
if present is outcompeted by patient DNA giving accurate
results.13 Such molecular typing methods are extremely
helpful for knowing actual antigen status of alloimmunized
patient.
Chronically transfused patients like Thalassemia, sickle
cell anemia, aplastic anemia, autoimmune hemolytic anemia,
etc. are often at a risk of developing delayed hemolytic
transfusion reaction (DHTR). In such multi-transfused
patients antigen matched donor units is recommended.
Previous studies have shown that methods like polymerase
chain reaction using sequence specific primers (PCR-SSP) for
blood group antigens (except ABO and RhD) prove successful
in determining the correct genotype of a multi-transfused
patient13 and thus can aid in efficient selection of the donor.
These techniques are much cheaper compared to serological
tests and also eliminate the ambiguities encountered with
mixed field agglutination. Molecular typing of antigens can
also help determine the additional blood group antigens to
which the patient may get sensitized. Blood samples from
fifty multi transfused thalassemia patients were studied by us
by hemagglutination and by PCR-SSP for common antigens
or genes in Rh, Kell, Kidd and Duffy blood group system.
Out of fifty thalassemic patients tested, the genotyping was
concordant with the serological red cell phenotype in only
16 cases for five antithetical antigen pairs in four blood group
systems (unpublished observation).
Antigen Profiling in DAT
Positive Cases
Serological typing of blood group antigens in DAT positive
red cells is considered invalid due to coating of red cells
by immunoglobulin molecules. Immunoglobulin removal
techniques used are not always effective and may destroy an
antigen of interest. DNA based methods can be used to know
the antigen profile of the patient and antigen matched blood
can be given so that the patient is not further alloimmunized.
This may circumvent the need for repeated auto-absorptions
to rule out underlying RBC alloantibody. Castilho L et al. (2001)
has shown the use of DNA based genotyping in management
of patient's with ''warm'' -induced hemolytic anemia.14
Differentiation between
Alloantibody and Autoantibody
Multi transfused patients are often at a risk of forming allo
and autoantibodies. These antibodies are identified in
patient's serum using reagent red cells panel. The allo or
auto specificity of the antibody can be determined by antigen
typing of patients red cells. However, presence of donor cells in
patient's circulation and antibody coated red cells invalidates
serological phenotyping of red cells. Molecular assays can
help determine the true antigen status of the patient and thus
differentiate between alloantibody and autoantibody.
Quality Control in Reagent Red Cells
The detection and identification of alloantibodies to blood
group antigens relies on the use of well characterized reagent
red blood cells. Group O RBCs from donors are extensively
tested for all common polymorphic antigens and some
low and high prevalence antigens and the phenotype is
determined by reactions with commercial polyclonal and
monoclonal antisera. Some antibodies react strongly with
RBCs carrying appropriate antigen in single dose, whereas
other antibodies will react only when RBCs carry a double
dose of the antigen (dosage effect). The true dosage of
antigens, i.e. zygosity predicated by serological testing can
be confirmed only by molecular methods. Fy(a-b+) are
considered as Fybhomozygous based on serological testing,
however, molecular testing may reveal heterozygosity for Fyx
allele present along with Fyb, leading to weakened expression
of Fybantigen even in absence of Fya.15 We have prepared
inhouse reagent red cell panel for identification of antibodies
by screening O group regular donors for clinically important
antigens and confirming the antigen status by DNA based
typing, to provides greater quality assurance.16
Screening for Rare Blood
Group Donors
Provision of low incidence antigen (like Jsa, V, VS, Goa)
negative units can be difficult in patients producing antibodies
against them. However, reagent red cell identification panels
are not typed for these antigens as reliable antisera are not
available. Patients with antibodies to high incidence antigens
require antigen negative units to prevent adverse transfusion
reactions. Obtaining an antigen negative donor unit in short
time is challenging due to shortage to reagents (e.g. Kp (b-),
Lu(b-), Yt(a-), etc.) forlarge scale screening of donors. Hence,
PCR based methods can be used to mass screen donors for
low and high incidence antigens thereby increasing antigen
negative inventories and improving patient care.17,18
Donors lacking a single common antigen (like h, Duffy,
Kell, Kidd, MNS, etc.) or combination of common antigens
are also considered rare. Our study of screening O group
regular donors for clinically important blood group antigens
have shown that, approximately 4.74% lacked combination
of common antigens of Duffy and Kidd blood group systems.
Some uncommon phenotypes negative for combination of
antigens were identified and used in preparation of reagent
panel cells.19 As the serological antisera for identifying
these antigens are costly, therefore only a small number of
donors are typed for few antigens, limiting antigen-negative
inventory. Thus, DNA-based screening programs can
provide a beneficial alternative. High throughput molecular
techniques make it possible to determine multiple RBC
antigens simultaneously. With regard to cost efficiency, many
transfusion centers opt for conventional multiplex PCR to
screen antigen negative donors.
 Chapter 77  Molecular Genotyping and its Applications to Transfusion Medicine  295
HPA, HLA and HNA Typing
DNA-based methods are being commonly used for typing of
human platelet antigens (HPA), human leucocyte antigens
(HLA) and human neutrophil antigens (HNAs). HPA typed
products are needed in cases of immunized patients and
neonatal alloimmune-mediated thrombocytopenic purpura.
Platelet donors can also be typed and called for donation in
case of any of the above conditions. The HPA typed panel of
donors (registry) is already in place in certain countries.
HLA matched donors are required for tissue and organ
transplants. A marrow registry of typed HLA donors is
beneficial. Antisera to HLA antigens are difficult to obtain
and involves tedious typing methods. High throughput assays
are now being developed for typing of HPA and HLA antigens.
HNA antigens and antibodies are known to play important
role in pathophysiology of various immune neutropenias
including transfusion related acute lung injury. Due to
difficulty in handling neutrophils and rarity of the availability
of antisera, most HNAs are typed using DNA based methods.
Limitations of DNA-based Methods
DNA-based methods have the fundamental weakness of not
detecting directly the presence of an antigen on the surface of
a red cell. Testing by DNA analyses has technical, medical, and
genetic pitfalls, and is also not approved by the FDA.20,21 From
a practical perspective, not all blood group polymorphisms
can be analyzed; for example, if a large number of alleles
encode one phenotype, or alleles with a large deletion, or a
hybrid alleles, or when the molecular basis is not yet known.
Additionally, there is a high probability that not all alleles in
all ethnic populations are known. There are many genetic
events that cause apparent discrepant results between
hemagglutination and DNA test results; the genotype is not
the phenotype (e.g. null phenotypes of Rh, Kell and Kidd
systems).22-25 Confirmation by hemagglutination of predicted
antigen negativity is recommended using a reagent antibody
if available, and/or by cross-matching. Detection of null
phenotypes is more easy and simpler by hemagglutination.
The molecular bases associated with a large number of
antigens have been reported. However, in many cases the
analysis has been restricted to a relatively small number of
people. This information is being applied to DNA typing
with the assumption that such analysis will correlate with
RBC antigen typing in all populations. Population studies
from a variety of ethnic backgrounds should be analyzed to
establish the correlation between genotype and the blood
group phenotype.26 Till then, caution should be xercised
when recommending clinical practice based on DNA typing
for blood group antigens.
Conclusion
Genotyping for RBC polymorphisms will play a more
important role in the routine work of blood establishments
in future. It will not replace serological methods for ABO and
RhD grouping, for antibody detection and identification but
should be used for minor antigens typing. In serologically
complex situations, genotyping will support decision making
and will contribute to increased quality of red cell reference
laboratory work. Genotyping will allow a wider spectrum
of antigens accessible for routine typing. Donors typed for
clinically important different blood group antigens will
create database for antigen negative inventory which will
be useful for providing antigen matched blood. This will in
turn enable electronic selection of units antigen matched
to recipients at multiple blood group loci, thus significantly
improving transfusion outcomes. Molecular genotyping
will complement antigen typing in cases where serology
faces difficulties (e.g. positive DAT) or in complex situations
(e.g. clarification of ABO and Rh variants). Thus molecular
genotyping, used as an adjunct to hemagglutination, will be
a powerful tool that would radically change approaches used
to support patients in their transfusion needs.
References
1. Storry JR, Castilho L, Daniels G, et al. International Society of
Blood Transfusion Working Party on red cell immunogenetics
and blood group terminology: Cancun Report (2012). Vox
Sang. 2014;107(1):90-6.
2. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Genomic
analysis of clinical samples with serologic ABO blood grouping
discrepancies: identification of 15 novel A and B subgroup
alleles. Blood. 2001;98:1585-93.
3. Kulkarni SS, Vasantha K, Gupte SC, Mohanty D, Ghosh K.
Potential of commercial anti-D reagents in the identification
of partial D variants in Indian population. Indian J Med Res.
2007;125(5):641-4.
4. Kulkarni S, Colah R, Gorakshakar A, et al. Frequency of partial
D in Western India. Trans Med. 2008;18(2):91-6.
5. Matheson KA, Denomme GA. Novel 3'Rhesus box sequences
confound RHD zygosity assignment. Transfusion. 2002;42(5):
645-50.
6. Kulkarni S, Vasantha K, Ghosh K. RHD zygosity by serological
and molecular methods in the donor population of Mumbai,
India. Vox Sang. 2012;100(suppl 1):217.
7. Perco P, Shao CP, Mayr WR, et al. Testing for the D zygosity with
three different methods revealed altered Rhesus boxes and a
new weak D type. Transfusion. 2003;43(3):335-9.
8. Chiu WK, Murphy M, Fidler C, et al. Determination of RhD
Zygosity: Comparison of a double amplification refractory
mutation system approach and a multiplex real-time quantitative
PCR approach. Clinical Chemistry. 2001;47(4):667-72.
9. Daniels G. Variants of RhD–current testing and clinical conse-
quences. BJH. 2013;161:461-70.
10. Moise KJJ. Management of rhesus alloimmunization in
pregnancy. Obstet Gynecol. 2008;112:164-76.
11. Mujezinovic F, Alfirevic Z. Procedure-related complications of
amniocentesis and chorionic villous sampling: a systematic
review. Obstet Gynecol. 2007;110:687-94.
12. Scheffer PG, van der Schoot CE, Page-Christiaens GC, et al.
Noninvasive fetal blood group genotyping of rhesus D, c, E and
of K in alloimmunised pregnant women: evaluation of a 7-year
clinical experience. BJOG. 2011;118(11):1340-8.
296  Section 4  Miscellaneous
13. Rozman P, Dovc T, Gassner C. Differentiation of autologous ABO,
RHD, RHCE, KEL, JK, and FY blood group genotypes by analysis
of peripheral blood samples of patients who have recently
received multiple transfusions. Transfusion. 2000;40(8):936-42.
14. Castilho L, Rodrigues A, Pellegrino Jr J, et al. Blood group geno-
typing for the management of patients with “warm”-induced
hemolytic anemia. Transfusion Clinique and Biologique.
2001;8(S):S166.
15. Storry JR, Olsson ML, Reid ME. Application of DNA analysis to
the quality assurance of reagent red blood cells. Transfusion.
2007;47(suppl 1):73-8.
16. Kulkarni S, Choudhary B, Gogri H, et al. Quality assurance of
reagent red blood cells by DNA analysis. Asian J Transfus Sci.
2014;8(suppl 1):6-25.
17. Reid ME. Applications and experience with PCR bases assays
to predict blood group antigens. Transfus Med Hemother.
2009;36(3):168-78.
18. Jungbauer C, Hobel CM, Schwartz DW, Mayr WR. High-
throughput multiplex PCR genotyping for 35 red blood cell
antigens in blood donors. Vox Sang. 2012;102(3):234-42.
19. Vasantha K, Kulkarni S, Ghosh K. Identification of donors
lacking a combination of clinically significant common blood
group antigens. Vox Sang. 2009;97(suppl 1):96.
20. Reid ME, Rios M. Applications of molecular genotyping to
immunohaematology. Br J Biomed Sci. 1999;56:145-52.
21. Reid ME. Applications of DNA-based assays in blood group
antigen and antibody identification. Transfusion. 2003;43:1748-
57.
22. Avent ND, Reid ME. The Rh blood group system: a review.
Blood. 2000;95:375-87.
23. Huang CH, Liu PZ, Cheng JG. Molecular biology and genetics
of the Rh blood group system. Semin Hematol. 2000;37:150-65.
24. Lee S, Russo DCW, Reiner AP, Lee JH, et al. Molecular
defects underlying the Kell null phenotype. J Biol Chem.
2001;276:27281-9.
25. Lucien N, Sidoux-Walter F, Olivès B, Moulds J, Le Pennec PY,
Cartron JP, Bailly P. Characterization of the gene encoding the
human Kidd blood group/urea transporter protein: evidence
for splice site mutations in Jknull individuals. J Biol Chem.
1998;273:12973-80.
26. Reid ME. Applications and experience with PCR bases assays
to predict blood group antigens. Transfus Med Hemother.
2009;36(3):168-78.
Rare Blood Group Registry
Swati Kulkarni
Blood group antibodies play an important role in transfusion
medicine during blood transfusion and pregnancy. Though
all antibodies are not clinically significant, many have the
potential to be. Importance of the system is based on whether
the antibodies can facilitate accelerated destruction of red
cells having the corresponding antigen. At present, 339
antigens have been discovered on human red cells of which
297 are clustered within 33 blood groups systems and the
remaining are accommodated in the Collections, 700 Series
of Low Incidence Antigens or the 901 Series of High Incidence
Antigens.1
Blood samples of donors and patients are not routinely
tested for antigens of other blood group system as the
antibodies against these antigens are not of regular
occurrence. As antibody screening and identification is not
performed in all blood banks in India, the problems come
to light when it is difficult to obtain compatible donors for
the patients. Even after the identification of the antibody/
antibodies, selection of blood is done by cross matching with
large number of donors as rare donor registry is not available
in our country. A systematic study of screening the regular
blood donor population for different blood group system
antigens, identifying rare donors negative for high frequency
antigens (HFA) and those who lack common antigens of
clinically significant blood group system will help in selecting
donors for patients with multiple antibodies. This registry will
have immense applicability across the country as the number
of multi-transfused patients are rising (Thalassemia, sickle
cell anemia, MDS, aplastic anemia, chronic renal failure,
etc.). We can also contribute significantly to World Health
Organization International rare donor panel.
Defining Rare Blood
The definition of rare blood group varies from country
to country.2 A donor who lacks HFA is considered to be
rare, which means the prevalence of such a donor in the
population is 1:1000 or less.3 The second category of rare
donors, are those who are negative for combination of HFA
78
(frequency varies from 1:200 to 1:1000). Individually some of
these antigens would not be rare, but combined phenotype
fits in above mentioned definition. The owner of this rare
phenotype is able to mount a clinically significant immune
response against the missing antigens. Thus a rare donor
can be a null phenotype, homozygote for an infrequent
antithetical antigen or combination of antigens. In some
countries all donors with phenotype with a frequency of less
than 1% of population is considered to be rare. Donor is also
considered rare if his/her red cell antigen profile differs from
majority of the population.
According to American Rare Donor Program, a donor
phenotype is considered rare if it meets one of the following
criteria: (1) Group O and Group A; R1R1, R2R2, R0R0, or
rr; and K:-1 and Fy(a-) or Fy(b-); and Jk(a-) or Jk(b-); and
S-or s-. (2) Group O and group A; R1R1, R2R2, or rr; and
K:-1; and Fy(a-b-). (3) All ABO groups; negative for high
incidence antigens (1:10,000) such as U, Jsb, Kpb, Yta or Ge2.4
According to French regulation, a blood donor is regarded as
rare if its prevalence is equal to or less than 1:250. However,
the whole phenotype of the donor is taken into account in
cryopreservation program. For example, Kell homozygous
(KK) donor is considered rare only if he is R1R1 or R2R2 or
rr with homozygous expression of Fya/Fyb, Jka/Jkb and S/s
antigens.5 Thus with the above variation in defining rare blood
groups, it is observed that when blood donor is not available
easily after cross-matching with ABO and RhD, compatible
donors can be called rare (mostly negative for single HFA
and their combination) and when compatible blood is found
after screening large number (more than 1000 donors) can be
called very rare donors.
International Rare Donor Program
Blood of rare blood groups is required rarely, but when
required National and International cooperation can ensure
that blood is made available to specified patients. The need
for a rare donor registry was noted at an early stage of blood
transfusion development. By 1959, American Association of
298  Section 4  Miscellaneous
Blood Banks had developed rare donor file and American
Red Cross, the rare donor registry.6 In 1965, the International
Society of Blood Transfusion (ISBT) formed the World Health
Organization International Donor Panel which is run by
International Blood Group Reference Laboratory (IBGRL).7
The ISBT working party on Rare Blood Donors was formed in
1986 with the following objective:8
• Developing guidelines on all matters related to the use,
labeling, testing and transport of rare blood
• Providing a resource for on going information on this topic
• Communicating with the WHO, IBGRL
• Upgrading international information on rare blood
programs
• Arrange many lectures and seminars on various aspects
of rare blood at national and international meetings to
increase the knowledge regarding problem of rare blood
supply.
The number of rare donors in International Donor Panel
had grown over the years and may be accessed by authorized
user via the web site http://www.bloodnet.nbs.nhs.uk/ibgrl.
The list continues to be compiled by IBGRL in Bristol, UK.9
Various strategies are adopted internationally to tackle this
important problem. The first involves setting up a Regional or
National Reference Laboratory, whose staff is suitably skilled
to carry out multiple and uncommon investigations on
large scale for screening rare donors and is able to perform
family studies in order to identify potential blood donors. The
second strategy is to set up registries of rare donors, in order
to ensure suitable transfusion support for the most critical
cases. These needs have been identified internationally and
over the years, numerous countries in Europe, America and
Asia developed extensive national programs for the supply
of rare blood. The programs were also supported by the
development of frozen rare RBCs bank.
National Status
Rare Blood groups may also vary from one country to another
and therefore blood type rare in one country may not be rare in
another. For example, Fy(a-b-) phenotype is virtually unknown
in the white population while this type reaches approximately
100% in some Black ethnic groups. Fy(a-b+) and D negative
is very rare in Chinese population.10 In our country, blood
donors and recipients of blood transfusion are tested routinely
for ABO and RhD blood groups, but antibody screening and
identification is not performed in majority of the blood banks.
The requirement for rare blood arises when blood samples
of the patient is not compatible with a number of donors.
Mostly screening for antibodies and their identification is
performed at this stage. Some big blood banks in major cities
in India perform tests for antibody identification, others
send to reference laboratory, while in remaining the blood is
transfused after cross matching with large number of donors
as antigen negative inventory of donors is not available. Also
the frequency of common antigens of clinically significant
blood group system is not known in various ethnic groups
in different parts of the country. Recently few studies on
limited number of donors are reported to find the frequency
of minor blood group antigens in Indian population,11,12 but
no systematic study to prepare rare blood group registry has
been carried out.13 Therefore, even when antibody/antibodies
are identified it is not possible to know how many donors
would have to be screened for getting a compatible blood
unit.
Bombay phenotype (Oh), a very rare blood group and In
a antigen of Indian blood group system was discovered at
National Institute of Immunohaematology (NIIH), Mumbai,
in 1952 and 1973 respectively.14,15 At our institute, we
maintain a registry of Bombay phenotype individuals whose
samples have been referred to us for investigations due to
problems in blood grouping and cross matching. The donors
are contacted for donation whenever there are requests. We
get request from various blood banks/hospitals from all over
the country and sometimes even from neighboring countries
for Oh blood units. New Oh donors can be identified by
performing family studies. The incidence of Oh phenotype
was found to be 1:7600 in Mumbai and 1:2500 in south west
part of Maharashtra among Maratha population.16 We detect
about ten cases of Oh phenotype every year at our reference
center at NIIH. But as the donor patient ratio is 1:4, it is not easy
to get a donor in time when in need. Other phenotypes like
D--/D--, In(a+b-) and Co (a-b-) are sporadically requested
in practice and we keep track of these patients and their
family members with these rare phenotypes as prospective
donors. Joshi and Vasantha have reported in detail the profile
rare blood groups reported from India.17 Rare blood group
phenotypes encountered at the NIIH are Bombay phenotype
(Oh), Para Bombay phenotype, Mg antigen, I-i- phenotype,
In(a+b-) Co(a-b-), D--/D--, Rh null and Rh phenotypes
like RzRZ, R1RZ, R2Rz, ryr, etc. Joshi (1992) has reported
screening of over 2000 random blood donors to provide two
units of In(a+b-) blood. Most often we were successful in our
approach to screen and find rare phenotype within the family
members of the proband.
Need for National Registry
Generally HFA do not cause any problems in transfusion
since the recipient is most likely also positive for the antigen.
The problem arises when a patient lacks one or two more of
these HFA and requires blood, or has antibodies to one or
more HFA. We are still dependent on cross matching with
large number of donors to find compatible antigen-negative
blood. To start screening donors after it is required, delays the
transfusion process as the likelihood of finding compatible
blood is less than 1 in 100 (depending on the antigen).
Consequently, this scenario usually has two outcomes unless
antigen-negative donors are known: to cancel the pending
operation or to delay the transfusion in order to obtain
appropriate compatible blood from other blood banks or
the WHO Rare Donor Registry, or from registry from other
countries which may take several days. The cryopreserved

unit obtained from International rare donor programs needs
to be washed and resuspended, and can in end leads to a blood
unit with less effect compared to a fresh blood unit. Seltsam
et al. reviewed current practice in Germany, Austria and
Switzerland and concluded that about one third of patients
with high frequency antibodies received unsatisfactory
transfusion therapy.18 Therefore rare donors should be
identified at national level and only for some very rare
phenotypes not available or screened in our population; help
should be taken from International rare donor programs. Also
the identification of rare donors negative for HFA and their
combination is essential as the frequency of these antigens
and antibodies produced by Indian patients will be different.
Our preliminary study of screening O group regular donors
have for clinically important blood group antigens have
shown that approx. 4.74% lacked combination of common
antigens of Duffy and Kidd blood group systems. Few donors
were very rare, i.e. they lacked Fya, Jka and s antigen with rare
Rh phenotype. Some uncommon phenotypes were identified
and used in preparation of reagent panel cells.19
Identification of Rare Donors
Identification of rare donors is critical factor in establishing
rare donor registry. The rare groups get identified during cross
matching, when compatible blood is not available, while
performing antibody screening on patient’s or donor’s serum
and during deliberate screening programs for detection of
rare groups. When a patient blood is not compatible with large
number of donors, or has multiple antibodies, the sample
should be sent to reference laboratory/regional transfusion
centers, so that further work up can be performed and rare
blood group identified can be included in registry.
Mass screening allows many new donors with rare
phenotypes to be identified. Antigen screening for absence
of HFA on mass scale begins with few antigens tested in
large number of donors. The donor lacking one or more HFA
is then tested for lack of other antigens that may constitute
a rare combination. As the discovery of number of blood
group systems and their antigens is increasing, the question
arises as to which antigens should the donors be typed
for? This depends on the rate of these antigens to induce
alloimmunization, their prevalence in discrete population
and clinical significance of alloantibody. As the allele
frequency can differ widely in some population, there are
special needs to screen RBC antigens in different regions of
the world. The data from of rare blood supplied in France and
Austria show the regional aspect in the need of rare blood and
indicate that screening for ‘most needed’ rare blood groups
differ from region to region.18, 20
Methods for typing blood group
types
Hemagglutination has been the gold standard for blood
grouping and antibody detection and identification. However,
Chapter 78  Rare Blood Group Registry  299
despite its ease of performance, and sensitivity and specificity
suitable for what we now consider to be optimal patient care,
hemagglutination-based determination of RBC phenotype
has its limitations. As the serological antisera for identifying
common antigens of clinically important blood group system
like Rh, Duffy, Kell, Kidd, MNS, etc. are costly, small number
of donors are typed for few antigens, limiting antigen-negative
or rare donor registries. The most important resource in
screening process is availability of reliable, potent antisera on
large scale and staff devoted to mass antigen screening. Some
RBC typing reagents are either not available or made from
saved patient’s plasma with the desired antibody specificity.
These typing reagents are human derived antibodies, may be
only weakly reactive, costly, difficult to obtain commercially
and not sufficient for large scale screening.
Over the past 20 years, the molecular basis of almost all
major blood group antigens have been determined, and it is
now possible to know the phenotypic expression of patients/
donors blood group by simple PCR methods. Blood group
genotyping is already a common practice in many blood
centers and larger Transfusion Services in western countries,
but only used in cases where serological typing is impossible
due to multiple transfusions or problematic serology.
Most blood group antigens result from single-nucleotide
polymorphism (SNP) giving rises to single amino acid
substitution which either inactivates the gene product or
changes its substrate specificity. These SNPs can be identified
at molecular level using various molecular techniques like
PCR-SSP, PCR-SSOP, RT-PCR, Sequencing, Pyrosequencing,
Microarrays, etc. Some of these high throughput molecular
techniques make it possible to determine multiple RBC
antigens simultaneously. For example, The Beadchip
(Bioarray Solutions, Warren, NJ), Genome Lab SNP Stream
(Beckman Coulter, Fullerton cA) and Blood Chip platform
(Progenika) have been used to genotype multiple blood
group antigens.21 In near future, donors and patients will
be genotyped on a mass scale using these technologies.
With regard to cost efficiency, many transfusion centers
opt for conventional multiplex PCR to screen HFA negative
donors. The Vienna Blood Center found that the cost of
supply of antigen-negative blood units for 2% of the blood
recipients was approximately 20% of the overall costs of
donor immunohematology by serological techniques. Their
calculations also revealed that donor antigen typing by DNA
screening was the cheapest method to increase antigen
negative database.22 Automated methods for mass screening
can reduce the technologists load and also use less amount
of reagent per test. The cost of these equipments, supplies,
maintenance and validation may prohibit the facility from
using this resource, even though it is available.
Future Directions
In India the aim should be to create a greater awareness of
the availability of rare donors. Also as the request for rare
blood to any center may be sudden and unexpected, there
300  Section 4  Miscellaneous
is needed to have the systems in place well before the event
occurs. A sufficient stock of pre-typed donors in the databases
enables a short response time for the provision of the blood
units requested and prevents acute on-demand typing. A
systematic screening for rare blood in India at various blood
banks in major cities in India and creating a database of the
rare donors as well as antigen negative inventory of clinically
important antigens (C, c, E, e, Fya, Fyb, Jka, Jkb, K, k, M, N, S,
s, etc.) present in Indian population is the need of the hour.
Some very rare donors like Bombay phenotype should
be cryopreserved so that they can be easily available and
transported in time. The Government should provide funds
for large scale screening and developing cryopreservation
facilities. This will lay foundation for the preparation of
database of rare blood groups for our country. India can also
actively contribute towards the International Donor Panel.23
With many major population movements throughout the
world and the potential consequent development of rare
antibodies the need for availability of rare blood is likely to
increase in future.
References
1. Storry JR, Castilho L, Daniels G, et al. International Society of
Blood Transfusion Working Party on red cell immunogenetics
and blood group terminology: Cancun Report (2012). Vox
Sang. 2014;107(1):90-6.
2. Nance ST. How to find, recruit and maintain rare blood donors.
Curr Opin Hematol. 2009;16(6):503-8.
3. Reesink HW, Engelfriet CP, Schennach H, et al. Donors with
rare pheno (geno) type. Vox Sang. 2008;95:236-53.
4. Flickinger C. In search of red blood cells for alloimmunized
patients with sickle cell disease. Immunohaematology. 2006;
22:136-42.
5. Peyrard T, Pham BN, Le Pennec PY, Rouger P. The rare
blood groups: a public health challenge. Transfus Clin Biol.
2008;15(3):109-19.
6. Mallory D, Malamut D, Sandler SG. A decade of rare blood
donor services in the United States (1981-1990). Vox Sang.
1992;63:186-91.
7. Mourant AE. The establishment of an international panel of
blood donors of rare types. Vox Sang. 1965;10:129-32.
8. Woodfield G. Rare Blood Donors: The past and the future. Vox
Sang. 2002;83(suppl 1):93-7.
9. Woodfield G, Poole J, Nance ST, Daniels G. A review of the ISBT
rare blood donor program. Immunohaematology. 2004;20:
244-8.
10. Beattie KM, Shafer AW. Broadening the base of a rare donor
program by targeting minority populations. Transfusion.
1986;26(5):401-4.
11. Makroo RN, Bhatia A, Gupta R, Phillip J. Prevalence of Rh,
Duffy, Kell, Kidd and MNSs blood group antigens in the Indian
blood donor population. Indian J Med Res. 2013;137:521-6.
12. Thakral B, Saluja K, Sharma R, Marwaha N. Phenotype
frequencies of blood group systems (Rh, Kell, Kidd, Duffy,
MNS, P, Lewis, and Lutheran) in north Indian blood donors.
Transfus Apher Sci. 2010;43:17-22.
13. Kaur R, Jain A. Rare blood donor programme in the country:
Right time to start. Asian J Trans Sci. 2012;6:1-2.
14. Bhende YM, Deshpande CK, Bhatia HM, Sanger R, Race RR,
Morgan WTJ, Watkins WM. A “new” blood group character
related to ABO system. Lancet. 1952;1:903.
15. Badakere SS, et al. Further observations on the Ina (Indian)
antigen in Indian populations. Vox Sang. 1974;26:400-1.
16. Bhatia HM, Sathe MS. Incidence of ‘Bombay’ (Oh) phenotype
and weaker variants of A and B antigen in Bombay (India). Vox
Sang. 1974;27:524-32.
17. Joshi SR, Vasantha K. A profile of rare blood in India and its
impact on transfusion services. Asian Journal of Transfusion
Science. 2012;6:42-3.
18. Selstam A, Wagner FF, Salamn A, Flegel WA. Antibodies to high
frequency antigens may decrease the quality of transfusion
support: an observational study. Transfusion. 2003;43:1563-6.
19. Vasantha K, Kulkarni S, Ghosh K. Identification of donors
lacking a combination of clinically significant common blood
group antigens. Vox Sang. 2009;97(suppl 1):96.
20. Reesink HW, Engelfriet CP, Schennach H, et al. Donors with
rare pheno (geno) type. Vox Sang. 2008;95:236–53.
21. Monteiro F, Tavares G, Ferreira M, et al. Technologies involved
in molecular blood group genotyping. Vox Sang. 2011;6:1-6.
22. Christ of Jungbauer Molecular Bases and Genotyping for Rare
Blood Types Transfus Med Hemother. 2009;36:213-8.
23. Woodfield G. Rare Blood Donors: The past and the future. Vox
Sang. 2002;83(suppl 1):93-7.
New Versus Old Blood

Rimpreet Singh Walia

79
Blood is a vital health care resource and red blood cells (RBCs)
are the most commonly transfused blood components.1
However, blood transfusion is a treatment with substantial
risks, associated costs and limited supplies.2 Therefore, it
has been a national goal that blood transfusion should be
safe and effective with adequate availability.3 Some of these
concerns regarding safety and adequacy of blood supply have
been addressed by the development of blood storage systems
that help in maintaining the effectiveness by preserving the
lifespan and function of red blood cells to the greatest extent
possible.1
Over the time, improvements in storage systems for RBCs
has increased the permissible length of storage from just 21
days with citrate-phosphate-dextrose (CPD) to 35 days with
citrate-phosphate-dextrose-adenine (CPDA) and blood Figure 1:  Advantages of using stored RBCs for transfusion
banks can, nowadays, store and supply RBC units to up to 42
days with the currently available additive solutions. The extent
of the length of storage of RBC units is based on the criteria even at these cold temperatures.5,6 Therefore, transfusion
that they can be stored as long as the average hemolysis is of a fresh RBC unit less than 5 days old from a donor who
below 1% and the recovery of RBCs 24 hours post transfusion was in the seronegative phase at the time of donation may
is higher than 75%.4 lead to transmission of syphilis in the recipient.7 Fourthly,
The increased length of storage time for RBCs has some storage reduces the chances of transfusion-associated graft-
advantages (Fig. 1). Firstly, blood is a precious commodity versus-host disease (TA-GVHD) in the recipients.4 Fresh
and world over there is a delicate balance between the blood predisposes patients to TA-GVHD probably because
growing demand and limited supply. Improved storage of it contains more number of viable lymphocytes. Storage
RBCs allows the accumulation and better management of decreases the number of viable lymphocytes8 and the
inventory4 which reduces wastage and is especially helpful expression of cell surface lymphocyte activation antigens.9
in life saving emergencies. It has been estimated that discard Therefore, TA-GVHD following transfusion of blood that is
rates were close to 30% when RBCs could only be stored for more than 4 days old is very rare.9,10
three weeks, whereas the current storage time ensures that On the other hand, several studies have reported that the
most of the blood banks are able to use more than 99% of increased length of storage of RBCs may be associated with
their RBC inventory. Secondly, longer storage provides ample the transfusion recipient’s increased morbidity, mortality
time for better processing, extensive testing and allows the and length of stay in the hospitalas compared to relatively
development of better quality controls. Thirdly, longer cold ‘Fresh Blood’.11
storage reduces potential transmission of syphilis through It is now well established that RBCs undergo several
blood transfusion.4 This is because, Treponema pallidum, the biochemical and physical changes during storage, which are
organism that causes syphilis, is unable to survive prolonged collectively referred to as “storage lesions”12 (Table 1). It has
cold storage at 4°C. However, it may live for 1 to 5 days been seen that lactic acid and protons, which are the products
302  Section 4  Miscellaneous
of glycolytic metabolism accumulate over time during RBC
storage.13 These protons lead to acidification, which alters
glycolysis resulting in a decrease in the concentration of 2,3-
diphosphoglycerate (2,3-DPG), which is typically gone by the
10th day of RBC storage, whereas adenosine triphosphate
(ATP) concentrations initially increase or are stable during
the first 2 to 4 weeks of storage with generally declining
concentrations thereafter.14 These changes are reported to
irreversibly alter RBC biological functions because of their
reduced deformability and increased osmotic fragility. As a
result of these changes the oxygen carrying capacity of the
RBCs decreases and the cells become rigid, resulting inan
impaired microcirculation.15-17
Longer storage of RBCs also leads to oxidative stress that
leads to lipid peroxidation, protein oxidation and reduced
integrity of the RBC membrane, resulting in the formation
of exocytic microvesicles, which have been linked to an
increased risk of post transfusion complications.18 Moreover,
there is loss of cation pumping with loss of intracellular
potassium; and rare episodes of bacterial contamination
and overgrowth.4 Furthermore, bio reactive substances
accumulate in the storage medium, which include
lysophospholipids that prime recipient neutrophils and have
been implicated in transfusion-related acute lung injury19
and accumulation of cytokines.20
Two mechanisms have been hypothesized to explain
the potential adverse effects of these storage lesions.21 One
hypothesis proposes that the RBC storage lesions cause
immunomodulatory and inflammatory complications
including changes in vasoregulation in transfusion recipients.
The second hypothesis suggests that susceptibility factors pre
dispose certain patient populations to the clinical pathologic
side effects of older RBC transfusion. Although RBC storage
lesions are well established, it remains to be determined
whether these changes lead to any functional complications
in the transfusion recipient.22
As mentioned above, storage leads to damage of
RBC membrane phospholipids with the elaboration of
lysophospholipids and these have been proposed to be
associated with increased rates of transfusion related acute
lung injury (TRALI) in transfusion recipients.23,24 However,
it has also been observed that rates of TRALI are markedly
reduced when only plasma from male donors is used for
Table1: RBC storage lesions
Physical changes Biochemical changes
Change in shape Decreased ATP
Decreased deformability Decreased 2,3-DPG
Increased osmotic fragility Increased extracellular potassium
Exocyticmicrovesicles Accumulation of bioreactive
substances
transfusion, therefore, the role of lysophospholipids in
causing TRALI has not been clearly established.25
Similarly, microvesicles shed from stored RBCs expose
negatively charged phospholipids on their surfaces that
are proinflammatory and procoagulant. Some studies have
suggested that transfusion of these RBCs may be associated
with increased inflammation, thrombosis and multiple organ
failure in critically ill patients; however, most of these studies
are confounded as the subjects are critically ill patients
receiving many kinds of therapy.4
It has also been postulated that stored RBCs lose their
ability to deliver oxygen due to loss of 2,3-DPG and impaired
microcirculation.26 However, the effect is small, and oxygen
delivery is reduced by about 15%.27 However, animal studies
failed to demonstrate any difference in critical oxygen delivery
between fresh and stored red blood cells.28
It has also been postulated that the temperature depend­
ent sodium potassium-dependent ATPase ‘pump’ on the RBC
surface29 is not able to overcome diffusive cation loss during
cold storage, thereby causing the intracellular potassium to
leak and accumulate in the extracellular medium at a rate of
about 1 mEq/l each day.4 The total extracellular potassium
load of older RBC units does not pose a problem during
transfusion except when such RBC units are infused through
central lines or into infants or used to prime cardiopulmonary
bypass or other high-flow devices,30 where the potassium
directly enters the central circulation and may cause cardiac
arrhythmias and even death. Therefore, relatively ‘fresh
blood’ which is less than 5–7 days old is given for neonatal
transfusions, exchange transfusions and transfusion in
cardiac surgeries.
Bacterial contamination is one of the rare but serious
adverse effects of prolonged storage of RBCs, although it
is more common in stored platelets that are stored at room
temperature, which favors bacterial growth. Most of the
bacteria, except Serratia marcescens, Yersinia enterocolitica,
and Aeromonas species do not survive in the refrigerated
temperature of 4–8°C used for RBC storage.31 The bacteria
which survive these cold temperature tend to grow slowly, and
it takes approximately 27 days for a single organism to grow to
108organisms and transfusion of such units may lead to sepsis
or endotoxin shock.4 However, bacterial contamination at the
time of blood collection can be minimized by proper donor
screening, thorough disinfection of the phlebotomy site and
use of diversion pouch.
Several studies have also reported that transfusion of older
units of RBCs was associated with an increase in length of
hospital stay, postoperative infections, prolonged mechanical
ventilation, multiple organ failure and mortality.32,33 However,
many authors do not report such association between RBC
storage age and adverse clinical outcomes33-36 and uncertainty
still remains on when “new” blood becomes “old” blood.12
Overall most of these studies have provided inconclusive
results and have a number of study design limitations. Many of

the studies are retrospective and observational in nature with
a small sample size and an inability to generalize the findings21
due to differential manufacturing practices and biased results
because sicker patients receive more transfusions and it is
difficult to establish whether the adverse outcome is a result
of transfusion only or the severity of the underlying disease.
As a result, the existing reports are inadequate and it is still
not possible to accurately conclude on the adverse outcomes
associated with the age of transfused RBCs.
Therefore, “the controversy between those who seek
longer red blood cell storage for logistical reasons and those
who have concerns about the safety and efficacy of stored
blood still continues”.4
Worldwide, all the blood banks follow the ‘first in and first
out’ policy which consists of transfusing the oldest compatible
and available RBC unit except in cases where the use of ‘fresh
blood’ is indicated. If it were assumed that transfusion of older
RBCs is associated with increased morbidity and mortality,
it would be prudent to transfuse fresh blood only. However,
a shorter outdate would have a major impact on the blood
supply especially in cases where rare groups are involved.
It was estimated that at over 1500 blood banks, 20% of RBC
units in stock were more than 28 days old37,38 and discarding
such units would further reduce the already limited blood
supply and create significant blood shortages. Moreover,
blood banks would require recruitment of new donors which
would involve additional risk, because first time donors are
more likely to carry transfusion transmissible infectious
diseases as compared to voluntary non-remunerated repeat
blood donors.7
Therefore, it is suggested that due to paucity of any
conclusive evidence, there is no need for any drastic
changes in transfusion practices and the blood banks should
determine the optimal age of red cells at issue in accordance
with the current medical and scientific evidence. However, the
blood transfusion services need to optimize their inventory
management according to clinical demand and whenever
possible, distribution of fresh blood should be encouraged
through implementation of ‘patient focused’ supply plan­
ning. For this, the blood transfusion services should devise
and employ statistical forecasting tools to better analyze the
supply trends and to set appropriate upper and lower limits
for red cell inventory. Moreover, emphasis should be laid on
the introduction of measures to reduce RBC storage lesions
like implementation of universal leucodepletion. In the
meantime, the blood transfusion services should also actively
engage with the broader clinical community to gather data
and conduct carefully designed, prospective controlled trials,
to determine whether there is any independent association
between the age of stored RBCs and transfusion related
complications and also to determine the optimal age of red
cells at the time of transfusion.39 The results obtained from
such trials should be compiled, statistically analyzed and
used to structure future guidelines regarding the use of ‘fresh
or old/stored blood’.
Chapter 79  New Versus Old Blood  303
References
1. Solheim BG, Hess JR. Chapter 4. Red cell metabolism and
preservation. Simon TL, Snyder EL, Solheim BJ, Stowell CP,
Strauss RG, Petrides M, (Eds). Rossi’s Principles of transfusion
medicine, 4th edn. Wiley-Blackwell; 2009. pp. 54-68.
2. Shander A, Gross I, Hill S, et al. New perspective on best
transfusion practices. Blood Transfus. DOI 10.2450/2012.
0195-12
3. Objective – 1. An Action Plan for Blood Safety. National AIDS
Control Organisation (NACO), Ministry of Health & Family
Welfare, Government of India, June 2007.
4. Zimrin AB, Hess JR. Current issues relating to the transfusion of
stored red blood cells. Vox Sang. 2009;96:93-103.
5. Van der Sluis JJ, Onvlee PC, Kothe FCHA, et al. Transfusion
Syphilis, Survival of Treponema pallidum in Donor Blood I.
Report of an Orientating Study. Vox Sang. 1984;47:197-204.
6. Van der Sluis JJ, Ten Kate FJW, Vuzevski VD, et al. Transfusion
syphilis, survival of Treponema pallidum in donor blood II.
Dose dependence of experimentally determined survival
times. Vox Sang. 1985;49:390-9.
7. Park AY, Brecher ME. Chapter 49. Bacterial Contamination
of Blood Products. In: Simon TL, Snyder EL, Solheim BJ,
Stowell CP, Strauss RG, Petrides M, (Eds). Rossi’s Principles of
Transfusion Medicine, 4th edn. Wiley-Blackwell; 2009. pp. 773-
90.
8. Qu L, Triulzi DJ, Rowe DT, et al. Stability of lymphocytes and
Epstein-Barr virus during red blood cell storage. Vox Sang.
2007;92:125-9.
9. Chang H, Voralia M, Bali M, et al. Irreversible loss of donor
blood leucocyte activation may explain a paucity of transfusion-
associated graft-versus-host disease from stored blood. Br J
Haematol. 2000;111:146-56.
10. Ohto H, Anderson KC. Survey of transfusion-associated graft-
versus-host disease in immunocompetent recipients. Transfus
Med Rev. 1996;10:31-43.
11. Marik PE, Corwin HL. Efficacy of red blood cell transfusion in
the critically ill: a systematic review of the literature. Crit Care
Med. 2008;36:2667-74.
12. Grazzini G, Vaglio S. Red blood cell storage lesion and adverse
clinical outcomes:post hoc ergo propter hoc? Blood Transfus.
2012;10(Suppl 2):s4-6.
13. Hess JR, Greenwalt TJ. Storage of red blood cells: New
approaches. Transfus Med Rev. 2002;16:283-95.
14. Korte D, Kleine M, Korsten HG, et al. Prolonged maintenance
of 2,3-diphosphoglycerate acid and adenosine triphosphate in
red blood cells during storage. Transfusion. 2008;48:1081-9.
15. Lacroix J, Tucci M. Impact clinique de la durée de conservation
des globules rouges avant transfusion. Transfusion Clinique et
Biologique. 2011;18:97-105.
16. Edgren G, Kamper-Jorgensen M, Eloranta S, et al. Duration
of red blood cell storage and survival of transfused patients.
Transfusion. 2010;50:1183-93.
17. D’Alessandro A, D’Amici GM, Vaglio S, et al. Time-course
investigation of SAGM-stored leukocyte-filtered red bood cell
concentrates: from metabolism to proteomics. Haematologica.
2012;97:2001-9.
18. Rinalducci S, D’Amici GM, Blasi B, et al. Peroxiredoxin-2 as
a candidate biomarker to test oxidative stress levels of stored
red blood cells under blood bank conditions. Transfusion.
2011;51:1439-49.
304  Section 4  Miscellaneous
19. Silliman CC, Moore EE, Kelher MR, et al. Identification of
lipids that accumulate during the routine storage of prestorage
leukoreduced red blood cells and cause acute lung injury.
Transfusion. 2011;51:2549-54.
20. Karam O, Tucci M, Toledano BJ, et al. Length of storage and
in vitro immunomodulation induced by prestorage leuko­
reduced red blood cells. Transfusion. 2009;49:2326-34.
21. Glynn SA. The red blood cell storage lesion: a method to the
madness. Transfusion. 2010;50:1164-9.
22. Whitsett C, Vaglio S, Grazzini G. Alternative blood products
and clinical needs in transfusion medicine. Stem Cells
International; Volume 2012, Article ID 639561, 14 pages
(doi:10.1155/2012/639561).
23. Silliman CC. The two-event model of transfusion-related
acute
lung injury. Crit Care Med. 2006;34:S124-S31.
24. Gajic O, Rana R, Winters JL, et al. Transfusion-related acute
lung injury in the critically ill: prospective nested case-control
study. Am J Respir Crit Care Med. 2007;176:886-91.
25. Eder AF, Herron R, Strupp A, et al. Transfusion-related acute
lung injury
surveillance (2003–2005) and the potential impact
of the selective use of plasma from male donors in the American
Red Cross. Transfusion. 2007;47:599-607.
26. Tinmouth A, Chin-Yee I. The clinical consequences of the
red 
cell storage lesion. Transfus Med Rev. 2001;15:91-107.
27. d’Almeida MS, Gray D, Martin C, et al. Effect of prophylactic
transfusion of stored RBCs on oxygen reserve in response to
acute isovolemic hemorrhage in a rodent model. Transfusion.
2001;41:950-6.
28. Torres Filho IP, Spiess BD, Pittman RN, et al. Experimental
analysis of critical oxygen delivery. Am J Physiol Heart Circ
Physiol. 2005;288:H1071–H9.
29. Hess JR. An update on solutions for red cell storage. Vox Sang.
2006;91:13-9.
30. Baz EM, Kanazi GE, Mahfouz RA, Obeid MY. An unusual case
of hyperkalaemia-induced cardiac arrest in a paediatric patient
during transfusion of a ‘fresh’ 6-day-old blood unit. Transfus
Med. 2002;12:383-6.
31. Brecher ME, Hay SN. Bacterial contamination of blood compo-
nents. Clin Microbiol Rev. 2005;18:195-204.
32. Lelubre C, Piagnerelli M, Vincent JL. Association between
duration of storage of transfused red blood cells and morbidity
and mortality in adult patients: myth or reality? Transfusion.
2009;49:1384-94.
33. Zimrin AB, Hess JR. Current issues relating to the transfusion of
stored red blood cells. Vox Sang. 2009;96:93-103.
34. Spinella PC, Doctor A, Blumberg N, et al. Does the storage
duration of blood products affect outcomes in critically ill
patients? Transfusion. 2011;51:1644-50.
35. van deWatering L. Red cell storage and prognosis. Vox Sang.
2011;100:36-45.
36. Cardo LJ, Hmel P, Wilder D. Stored packed red blood cells
contain a procoagulant phospholipid reducible by leuko­
depletion filters and washing. Transfus Apher Sci. 2008;38:141-
7.
37. CCBC. FDA committee endorses education and research to
combat rare bacterial reaction: Rejects operational changes for
now. CCBC Newsletter. 1991;10:1-4.
38. AABB. FDA blood products advisory committee supports
educational efforts on Yersinia enterocolitica. Blood Bank
Week. 1991;20:1-3.
39. Australian Red Cross—Blood Service Policy on “The Age of Red
Cells”. http://cdm16691.contentdm.oclc.org/cdm/singleitem/
collection/p16691coll1/id/23/rec/1.
Importance of Safe Blood
Administration Practices
Sharad Jain
“Right blood to the right person at right time in right quantity”.
Today is commonly talked about in relation to blood safety.
Transfusion services are handled by large group of technical
persons and series of instruments which plays an important
role hence system needs to be evaluated for safety purposes.
Blood transfusion is definitely quite safe due to several
innovations, advancement in technology, knowledge and
skills. Blood transfusion saves life, and improves quality of
life in large number of critical conditions. It is highly risky like
other intravenous drugs.
Serious hazards of transfusion (SHOT) are a professionally
reporting scheme, which collects data on the serious
consequences of the transfusion of blood components in
order:
• To educate users for transfusion hazards and their
prevention
• To improve the standards of hospital transfusion practices.
• To form the policy in transfusion services and to aid
clinical guidelines on the use of blood components.
SHOT assess blood transfusion staff for their competency
qualification, experience, making records, keeping records,
recorded justified reasons for transfusion, procedures
followed during blood transfusion, during transfusion
reaction and several other aspects.
SHOT survey of primary to tertiary care hospital, nursing
homes and blood banks, reviewed overall transfusion, SHOT
reports show several pit falls. Blood safety is a vital issue needs
realization, understanding and immediate attention. Safe
blood transfusion is only possible if procedures are followed
rigidly as per SOPs.
Despite several initiatives transfusion errors continues
to occur, as reported during transfusion practices by audit
worldwide.
Hospital clinical staff is responsible for: reception of blood
and blood products from the blood bank, correct storage in
wards, operating rooms and other clinical areas, monitoring,
operation of blood warmers and safe transfusion of blood and
blood products.
80
Blood component storage: Whole blood and red cells must
be stored at a temperature of +2°C to +6°C. Whole blood and
red cells never freeze. Never use domestic refrigerator for
blood storage. Blood storage cabinets are essential for blood
storage.
Fresh frozen plasma (FFP): The plasma which has been
separated within 6–8 hours and has been rapidly frozen to–
20°C or colder.
Cryoprecipitate is the cold-insoluble jelly like portion of
plasma remaining after FFP has been thawed. The optimal
storage temperature is –40°C.
Platelet concentrates: Manual and automated methods are
used in preparation of platelet. Concentrates must be kept at
a temperature between +20°C and +24°C in a platelet agitator.
Never refrigerate and keep idle.
Inspection of blood components: Before issue and transpor-
tation of components from the blood bank, inspection for any
evidence of deterioration, leakage and damage. Possibility of
bacterial contamination could cause severe or fatal reaction
if transfused. Each pack should be checked for hemolysis and
contamination during collection for transfusion.
Warming blood: Nurses and doctors prefer “warm up” blood
before transfusion. No warming required if administered
at a slow speed. Warming is justified if recipient receiving
large volumes of blood and component in very short time. It
prevents cardiac arrhythmia. Blood should be warmed in a
specified blood warmer.
Fresh frozen plasma should be thawed in the blood bank
water-bath at a temperature between +30°C and +37°C. The
plasma unit should be kept upright and, inside another
plastic bag to prevent contamination.
Fresh frozen plasma should be infused within 30 minutes
of thawing. If not immediately required can be stored in a
refrigerator at a +2°C to +6°C and transfused within 24 hours.
Blood and blood components are to be collected from
blood bank only for immediate transfusion within 30 minutes.
306  Section 4  Miscellaneous
Normally avoid return of blood to blood bank. If at all is
essential, return unopened bag within 30 minutes. In case of
return checklist is followed in blood bank to decide whether
it should be put back into stock or discarded. Bag checked
for temperature by folding the unit around thermometer.
Let blood settle in the refrigerator look for hemolysis or any
evidence of deterioration in the plasma and red cells.
SHOT analysis revealed errors during Blood transfusion
mainly are:
Blood sample collected from wrong patient.
Patient details were recorded incorrectly on blood sample
label or the blood request form.
Incorrect unit was collected from the blood storage
refrigerator.
Final identity check at the patient’s bedside, prior to
transfusion, was omitted or performed incorrectly.
“Only staffs who are trained and competent should
participate in the blood transfusion process.”
It is commonly observed that nurses and midwives
regularly take responsibility for completing the request form,
taking a blood sample for pre-transfusion testing, collecting,
administrating blood components and monitoring the
patient during the transfusion.
“The decision for blood transfusion must be based on
a thorough clinical assessment. The decision to transfuse
specific components should be documented in the patients’
clinical records.”
Informed written consent from patient: It is legal, ethical
and valid document. Consent must be obtained before blood
transfusion. The potential risks, benefits must be explained.
Valid consent is recorded in the patient’s clinical records
before transfusing blood component.
A patient identification band must be worn by all patients
receiving blood transfusion. Their first name, surname and
date of birth, IPD or OPD as check before collecting blood
sample.
Collect the required amount of blood into the appropriate
sample tube. Label immediately after collection before
leaving patient and sign the sample tube. Sample tube and
request form details must correspond. Collect only one
patient sample at a time and never pre-label the sample tube.
Prior to collecting the blood component ensures patient
is wearing an identification band, patient consent and
indication for transfusion are recorded in the patient’s
clinical record. Suitably trained and competent staff must be
available for transfusion. Patient’s baseline pulse rate, blood
pressure, temperature and respiratory rate checked and
recorded in a patient clinical record.
Ensure that every blood component collected is checked
against the patient’s minimum identification along with
blood component form, patient compatibility label showing
expiry date on the blood component with patient’s wristband.
Collect one unit at a time except in a massive transfusion.
Document the removal of the blood component by putting
the date, time and signature.
Preadministration procedure: Blood transfusion should be
started as soon as possible after blood component collected
explaining patient about Blood transfusion. Use I/V cannula
from 14 G to 24 G depending transfusion rate planned using
170–200 um pore size filter. No drugs to be added in blood
bag. No dextrose, plasma expanders to be given along with
blood transfusion.
Before each unit record baseline parameters, component
details on tag with expiry date, visual inspection for any signs
of discoloration, clumping or leaks.
Blood components must only be administered by a
registered health care professional who is trained and
competent.
The final administration check must be conducted bedside
by a trained professional administering component.
Repeat identification check details on the patient’s
identification band match details with compatibility label
attached to the blood component and transfusion form with
each component finally for surety.
Patient monitoring: Close observations during transfusion
should be undertaken and documented for every unit
transfused.
Good record keeping is an integral part of blood safety
practice, following FDA guidelines is essential for safe and
effective care.
Patient during transfusion must be observed from the
cabin, look for vague symptoms of transfusion reaction,
irritable, shivering, and breathless. Check and record
baseline parameters at 15 minute interval. Adjust initial
transfusion rate 8–10 drops per minute for first 30 minute,
later increase the rate. Record transfusion notes from starting
to end including post transfusion base line.
In any suspected transfusion reaction, first step is to
stop transfusion and immediately inform medical doctor.
Assure and explain patient and their relatives to relieve panic
situation. Assess patient’s airway, breathing and circulation
(ABC). If life-threatening reaction is anticipated, call for
resuscitation team. Maintain venous line patent with 0.9%
sodium chloride. Record the patient’s pulse, temperature,
respiratory rate, blood pressure, urinary output including
color for hemoglobinuria. Urgent evaluation of the blood
required at blood bank, microbiology for assessment of
contamination, hematological, biochemical parameters of
recipient. Patient needs a close observation and record vitals
at regular interval. If no improvement in clinical condition
found, transfer immediately to intensive care unit.
Check the core identification between the patient and
blood component compatibility label. Inspect the component
for unusual clumps, particulate matter or discoloration.
 Chapter 80  Importance of Safe Blood Administration Practices  307
Inform the hospital transfusion laboratory; return the unit
and administration set to the transfusion laboratory. Record
the adverse event in the patient’s clinical record.
The safety of blood transfusion is lying in multiple
hands at different levels. The key principles for blood safety
are based on competency, knowledge, experience and
dedication of staff to work. Automation prevents several
clerical errors. Care is to be taken in identification of recipient
from collection of sample to transfusion. Rationale blood
component, its identification, visual quality check including
expiry is essential. Keep close observation of recipient during
transfusion. Finally at every step documentation and record
keeping is a mark of skill and safe transfusion practices.
Suggested Reading
1. Emmanuel JC. The clinical use of blood. Handbook. WHO,
Blood Transfusion Safety, Geneva, 2001.
Strategies to Reduce
Unnecessary Transfusions
Hemchandra Pandey, Rajendra K Chaudhary
Providing safe and adequate blood has been integral
part of every country’s national health care policy and
infrastructure. Blood transfusions no doubt help in saving
lives but there is remarkably little evidence from randomized
controlled trials on what types of patients benefit from blood
transfusions, what blood products should be transfused,
and how much patients should receive. Due to lack of well
framed policies and guidelines, majority of patients get
unnecessary transfusions either in terms of over-transfusion
or inappropriate components transfused.
These unnecessary transfusions put the patients to a
number of risks including the risk of transfusion transmitted
infections, alloimmunization, hemolytic transfusion rea­
ctions, a higher incidence of postoperative infections and
various other known and unknown risks.1 Some of these risks
increase as the number of transfusions increase. Moreover,
unnecessary transfusions also increase the burden on blood
transfusion services in terms of cost as well as reduction of the
precious blood units from inventory in situations of already
scarce blood supply.
Blood transfusion services thus need to devise various
strategies to identify patients at risk for transfusions and
provide a standardized procedure for reducing or eliminating
the need of transfusion. These strategies start at the hospital
itself from the clinician attending the patient and spread to
cover the whole nation.
The first strategy is evidence-based blood use strategy. The
most important requirement to implement this strategy is the
presence of evidence based guidelines regarding the triggers
for transfusion. Traditionally, a hemoglobin measurement
of 10 g/dL was considered to be an appropriate transfusion
threshold, based on studies of rheology and oxygen delivery,
rather than outcome data. Current guidelines suggest it is safe
to lower this threshold to 8 g/dL, even in patients with cardio-
respiratory disease.2 An adoption of this evidence-based
blood use would reduce the unnecessary transfusions.
The second strategy is clinical strategy which is based
on application of evidence-based medical and surgical
concepts designed to optimize the patient’s hemoglobin,
81
minimize blood loss and bleeding and optimize the patient’s
physiology to tolerate anemia. This multidisciplinary strategy
has been named patient blood management and it is highly
individualized. It covers preoperative, intraoperative and
postoperative phases of patient’s blood management. In
preoperative period patients risk factors for bleeding during
surgery are identified and anemia is corrected with the use
of Hematinics and erythropoietin. During the intraoperative
phase various anesthetic and surgical techniques such as
proper patient positioning, controlled hypotension, use of
minimally invasive surgeries, use of cell salvage, etc. are used
to limit the blood loss. Similarly the use of Hematinics and
a restrictive transfusion therapy in the postoperative phase
help in reducing the unnecessary transfusions.2 Use of other
alternatives such as fibrin gel, platelet gel and antifibrinolytics
could be a part of this strategy.
Though the above two strategies if adopted properly will
reduce the unnecessary transfusion to a minimum, yet this
is not so as there is variability among various clinicians
regarding the use of blood transfusions as highlighted by
various studies.3 The main reason behind this practice
variation among clinicians is slow adoption of newer practices
or unfamiliarity with the newer practices. Thus a third strategy
targeting clinicians and other clinical staff involved in blood
transfusion becomes necessary. It is an education based
strategy wherein the blood transfusion knowledge is updated
at regular intervals by the use of Hospital wide educational
program, CMEs, panel discussions, etc. Such education based
interventions have proven to reduce the rate of unnecessary
transfusions in various studies.4-7
On the similar lines is the audit based strategy wherein
blood utilization practices could be analyzed and the clinician
made aware of the inappropriate transfusion practices.
These have the advantage of refreshing the knowledge of
the clinician in addition to pointing out the deviation from
guidelines.8-11
Though the above strategies are effective in reducing
transfusions but it is difficult to implement them at regular
intervals. Here comes the role of hospital transfusion
 Chapter 81  Strategies to Reduce Unnecessary Transfusions  309
committees which devise and implement the blood
transfusion policies, prepare educational programmes,
conduct regular audits and disseminate the guidelines relevant
to their center. A hospital transfusion committee provides a
mean to implement the above strategies at hospital level and
have been proven to reduce the unnecessary transfusion as
evident by various studies.12,13 Thus appropriate steps should
be taken to create hospital transfusion committees.14
In addition a national strategy to check the overall
progression of strategies at local level should be done. This
strategy involves creation of a hemovigilance network
wherein various aspects of blood transfusion are reported
and guidelines based on newer evidence are prepared.
This network also monitors the blood transfusion practices
and based of inappropriate practices formulates the new
practices.
Thus no single strategy could help in reducing the
unnecessary transfusions. A multiapproach strategy with
involvement of all right from the clinician to the hospital
to the national health agencies is required for reducing the
burden of unnecessary transfusions and saving this precious
resource so that it is available to a needy person in time.
References
1. Theusinger OM, Felix C, Spahn DR. Strategies to reduce the
use of blood products:a European perspective. Curr Opin
Anesthesiol. 2012;25:59-65.
2. Taylor SE, Cross MH. Clinical strategies to avoidblood
transfusion. Anaesthesia and Intensive Care Medicine. 14:2.
3. Thurer RL, Lambert C, Parce P, et al. Variability in transfusion
practice—beyond cardiac surgery. Transfusion. 2012;52
(suppl):121A.
4. Rehm JP, Otto PS, West WW, et al. Hospital wide educational
program decreases red blood cell transfusions. J Surg Res.
1998;75:183-6.
5. Morrison JC, Sumrall DD, Chevalier SP, et al. The effect of
provider education on blood utilization practices. Am J Obstet
Gynecol. 1993;169:1240-5.
6. Ayoub MM, Clark JA. Reduction of fresh frozen plasma use
with a simple education program. Am Surg. 1989;55:563-5.
7. Handler S. Does continuing medical education affect medical
care? A study of improved transfusion practices. Minn Med.
1983;66:167-80.
8. Khan FAH, Kamal RS. Improvement in intraoperative fresh
frozen plasma transfusion practice—impact of medical audits
and provider education. J Pak Med Assoc. 2000;50:253-6.
9. Toy PT. Effectiveness of transfusion audits and practice
guidelines. Arch Pathol Lab Med. 1994;118:435-7.
10. Kakkar N, Kaur R, Dhanoa J. Improvement in fresh frozen
plasma transfusion practice: results of an outcome audit.
Transfus Med. 2004;14:231-5.
11. Garrioch M, Sandbach J, Pirie E, et al. Reducing red cell
transfusion by audit, education and a new guideline in a large
teaching hospital. Transfus Med. 2004;14(1):25-31.
12. Torella F, Haynes SL, Bennett J, et al. Can hospital transfusion
committees change transfusion practice? JR Soc Med. 2002;
95(9):450-2.
13. Calder L, Woodfield G. The hospital transfusion committee:
a step towards improved quality assurance. NZ Med J.
1991;104(921):427-9.
14. Haynes SL, Torella F. The role of hospital transfusion
committees in blood product conservation. Transfus Med Rev.
2004;18(2):93-104.
 Index
Page numbers followed by f refer to figure and t refer to table.

A Antenatal testing and HDN status in after first centrifugation, layers of 198f
developing countries 62 and blood components, discarding of
Abdominal surgery, previous 90
Antibody 245
ABO
depletion 83 bank accreditation 273
antibodies 81
mediated rejection 78, 82 banks/centres, types of 273
antigens 81
severe 78 cell 190
blood group discrepancies 292
screening, automated 288 growth factors 31
blood-type selection 92
Antifibrinolytics 91 coagulation tests disturbance 18
desensitization 78
Antigen profiling in DAT positive cases 294 collection 248
grouping 92
hemolytic disease of newborn 69 Anti-platelet antibodies 158 component 92, 129
incompatibility Antithymocyte globulins 78 inspection of 305
in BMT 73 Apheresis in donor, adverse effects of 124 irradiated 93
major 73 Aplastic anemia 147, 154, 297 selection of 68
plus minor 73 acquired 3, 152 storage 305
minor 73 congenital 3 therapy 103
transfusions 136 inherited 3 conservation
minor 93 APPT 159 during cardiac surgery 46
ABOI-KT, adverse effects of 85 Aprotinin 30, 164 strategies 40, 57
Accreditation, scope of 273 Arteriovenous malformations 57 disease 182
Acidosis 98 Arthropathy 150 donation
Acquired immunodeficiency syndrome Autoimmune ethics for 281
(AIDS) 264 hemolytic anemia 7, 8-10, 292
gift 282
Adeno associated viruses 215 thrombocytopenia 120
donors
Adenoviruses 215 Autologous
for infectious agents 264
Adult stem cells 184 blood 92
screening 226
Agglutination, grading of 286f donation 40
establishments, ethical issues related to
Agranulocytosis 150 options 40
283
Aldehyde dehydrogenase rich bright cells transfusion 35
film examination 160
(ALDHBR) 195 Auto-recognition 76
for transfusion, selection of 7
Allergic
blood transfusion reactions 209 B group 288, 290f
antigens, typing of 293
reactions 255 Babesia protozoa 138
types 299
type reaction 5 Babesiosis 138
in transfusion practice, role of whole 35
Alloantibody and autoantibody, Bacterial contamination 256, 261
lactate dehydrogenase, elevated 18
differentiation between 294 in blood component 237
loss 91, 102
Allogeneic red blood cell transfusion 46 Bacterial detection
causes of massive 102
in cardiac surgery, associated with 46 direct methods of 238
Allograft rejection 76 massive 102
in blood components 236
Alloimmunization 257 new versus old 301
Bacterial peptidoglycan chromogenic
Alzheimer’s disease 181 RCC seropositivity, reasons for discarding
immunoassay 239
American Society for Apheresis 88 Barrier filtration 222 whole 247t
Amputation free survival 196 B-cell depletion 82 safety 219
Anaphylactic reaction 255 Bernard Soulier syndrome 109 initiatives 261
Anaphylactoid reaction 255 Beta-cell dysfunction 190 sparing surgical techniques 40
Anemia 15 Beta-thalassemia 214 system, intercept 242
correction 40 Bilateral total knee arthroplasty 49 transfusion 20, 21, 30, 144, 206, 281
in cancer patients, management of 15 Bleeding disorders 157 ethics for 281
of chronic renal failure 31 acquired 157 policies, implement 309
secondary to inherited 157 services (BTS) 279
chronic disorders 31 Bleeding time 158, 160 type kidney transplantation 81
zidovudine therapy 31 Blood 36 units, expiry of 250
Angina/ECG changes of ischemia 3 administration practices, importance of units, management of 250
Antenatal screening of thalassemia 65 safe 305 warming methods 97
312  Transfusion Update
Bone marrow 158, 207 Citrate-phosphate-dextrose (CPD) 301 Dermatology 199, 200f
failure 127 adenine (CPDA) 301 Dermatomyositis 134
acquired 3 Clot retraction test 158 Desferrioxamine 149
mononuclear cells (BMMNCs) 185 Clotting factor concentrate 168 Desmopressin 164
transplant (BMT) 141, 179 Clotting time 158 Diabetes 189
Bone morphogenetic protein 198 CMV transmission and leucocytes 224 type 1 189
Brain stem cell 190 Coagulation protein, deficiency of 157 Diabetic
Breath, shortness of 3 Coagulopathy in liver diseases, ketoacidosis 120
management of 162 wound healing 200f
C Cold Discarding FFP, reasons for 249t
Cancer 199 ischemia time 90 Disease, type of 189
surveillance 212 liquid-stored platelets 33 Disseminated intravascular coagulation 13,
Cardiac Column agglutination technique (CAT) 285 30, 56, 127
arrhythmias and failure 145 Combination therapy 152 DNA-based methods, limitations of 295
disease 256 Compatibility testing 140 Donor 67
iron, MRI of 148 Compatible blood components, selection cell cytokines 224
stem cells, resident 184 of 92 confidentiality 282
Cardiopulmonary Complete blood count 17, 18 deferral 282
bypass 44, 45 Congenital amegakaryocytic derived antibodies 93
surgery 127 thrombocytopenia 3, 17 ethical issues related to 282
disease 256 Connective tissue disorder 31 identification of rare 299
Cardiothoracic surgery 200f Cord blood platelet, dose of
Cardiovascular banking 204 random 124
complications 207 transplantation 204 single 125
disease 181 in India 205 platelets, advantages of single 125
in thalassemia in India 204 recruitment 248
surgery 36
Core rewarming, active 97 selection 129
Cataract 180
Coronary specific antibody 79
Cell
artery bypass grafting, primary 44 specific transfusion 77
adhesion 222
heart disease 185 Drug 3
aplasia, pure red 207
intervention 20 induced thrombocytopenia 120
contemplated for cellular regenerative
Cosmetic ophthalmic surgery 200f immune 17
therapy, types of 184
Critical limb ischemia (CLI) 194 Dyserythropoietic anemias, congenital 3
salvage 91
Crosslinked hemoglobin 31 Dyskeratosis congenita 3
stress 212
Cryoprecipitate 104, 164, 174
therapy 155
for neonatal transfusions 69
E
types of 184, 187
utilization guidelines, evidence-based Embryonic stem cell 184
Cellular
271 Endothelial
blood components, irradiation of 67
Culture-derived platelets 33 cell 198
therapies 177
Cyanoacrylates 164 progenitor cells (EPC) 184, 185
Central venous pressure 91
Cytapheresis 135 End-stage kidney disease 81
Centrifugation and buffy coat removal 223
Cytokine transfer/infusion, passive 224 Enhanced bacterial detection system 238
Cerebral
Cytomegalovirus 67 Enzyme
blood flow 26
Cytotoxicity, mechanism of 211 immunoassays 264, 266
ischemia, delayed 26 linked immunosorbent assays (ELISA)
malaria 136
Cerebrovascular surgery 56 D 230, 266
Epidermal growth factor (EGF) 202
Cerus 242 Danazol 30 Epoetin and darbepoetin in adults,
Challenges in gene targeting in human stem D-dimer test 158 guidelines for 16
cells 217 Deferasirox 144, 151 Epsilon aminocaproic acid 30, 91, 164
Chelation therapy 144 Deferiprone 150 Erythrocytapheresis 135
Chemically modified hemoglobin solutions Deferoxamine 144 Erythrocyte-magnetized technology 289
5 Deglycerolization 223 Erythroid colony forming units 31
Chemiluminescence 264 Del phenotypes, identification of 293 Erythropoiesis stimulating agents (ESAs)
Chemotherapeutic agents 18t Dendritic cells 213 38, 206
Chest syndrome, acute 154 Dengue effects of 206
Child-Turcotte-Pugh Score 89 fever 36, 110 versus red cell transfusion 206
Cholelithiasis 145 hemorrhagic fever 110 Erythropoietin 21
Cilostazole 194 infection, grading severity of 111t Escherichia coli 256
Citrate phosphate dextrose adenine (CPDA) shock syndrome 110 Esthetic surgery 199
198 Dentistry 200f External quality assessment scheme (EQAS)
Citrate toxicity 256 Deoxyribonucleic acid (DNA) 226 280
 Index  313

External rewarming, active 96 potential of 214 Hemorrhage 110

Extracorporeal photopheresis 88 prerequisite for 215 and massive transfusion, coagulopathy of
Genetic 96
F disease, therapy for 182 Hemorrhagic shock, physiology of 95
Familial thrombocytopenis-leukemia therapies 155 Hemostasis
syndrome 17 Germline gene therapy 214 defects in primary 162
Family study for genetic counseling 141 Glanzmann’s thrombasthenia 109 defects in secondary 162
Fanconi’s anemia 3, 147, 204 Global seroprevalence 232 in liver disease, secondary 163f
Febrile nonhemolytic transfusion reaction Globin gene therapy for treatment of Heparin-induced thrombocytopenia 17,
(FNHTR) 25, 209, 221, 223, 224, 254, beta thalassemia 216 118, 122
270 sickle cell anemia 216 Hepatitis
Febrile transfusion reaction 5 Graft function, delayed 91 B
Fetal and neonatal alloimmune Graft versus host disease (G vs HD) 3, 91, infection 232
thrombocytopenia 116 93, 180, 199, 221 vaccination 141
Fetal blood sampling 63 Graft-recipient body weight ratio 90 virus 230, 232, 265
Fever 252 Granulocyte assays, evolution of 265
FFP and SD plasma, difference between colony stimulating factor 31 C virus 230
171t for neonatal transfusions 70 assays, evolution of 264
Fibrin glue 164 transfusion 4 Hepatocellular carcinoma 91
Fibrinogen 104, 271 indications of 5 Hereditary hemochromatosis 136
assay 160 Growth retardation 150 High frequency antigens (HFA) 297
coated albumin microcapsules 33 Guillain-Barré syndrome 134 High performance liquid chromatography
deficiency 65
acquired 174 H High quality blood products, availability
congenital 174 of 208
Hemoglobin, stroma free 31
Histocompatibility complex, major 71
Fibrinolysis 163 Hemorrhage, acute 35
Hit, pathophysiology of 119f
risk of 172 Hair follicle viability 202
HLA
Fibroblast growth factor-2 (FGF-2) 202 Harmonic scalpel 40, 40f
alloimmunization 224
Filtration HBV infection 233
function of 71
and leucodepletion, effects of 224 Health technology assessment (HTA) 284
typing 71
mechanism of 222 Heart failure 185
Homologous recombination 217
Fluid warmer technology 97 Hematological parameters, preoperative 90
Human
Fluorescence activated cell sorting analysis Hematopoietic adult bone marrow derived stem cells
239 cells 185 185
Fluorescentprotein 185 growth factors 31 erythropoietin 31
Folic acid 144 stem cell 71, 190, 216 immunodeficiency virus 230
Freezing 223 transplantation 71 assays, evolution of 265
Fresh frozen plasma (FFP) 95, 103, 123, transplantation (HSCT) 179 leukocyte antigens (HLA) 179, 212, 295
164, 305 types of 179t neutrophil antigens (HNAs) 4, 25, 76, 78,
cryoprecipitate 114 Hemodilution techniques 52 81, 141, 295
for neonatal transfusions 69 Hemoglobin platelet antigens (HPA) 116, 295
Frozen plasma utilization guidelines, based oxygen carriers 31 Humoral rejection 77
evidence-based fresh 271 and red blood cells, comparison Hyperacute rejection 76
Frozen platelets 33 between 32t Hyperglycemia 145
Frozen thawed red cell 4 concentration 37 Hypertension 194, 207
measurement 308 Hypocalcemia 145
G Hemolytic Hypocoagulability 162
Gastrointestinal adverse effects: 151 crisis 154 Hypogonadotropic hypogonadism 145
Gel based technology 285 disease of Hypoparathyroidism 145
Gel technology fetus 62 Hyporeninemic hypoaldosteronism 207
advantages of 286 newborn 62, 138 Hypothermia 96, 256
applications of 286 severe 257 Hypothyroidism 145
principle of 285 reactions, acute 208
Gel test column agglutination technology transfusion reaction (HTR) 5, 209, 252, I
285f 270 Idiopathic thrombocytopenic purpura 3, 9
Gene delayed 5, 257, 294 chronic 30
delivery systems 215 immediate 252 ID-NAT versus MP-NAT 230
therapy 155, 182 uremic syndrome 18 Immune
future of 217 Hemophilia A 174 causes, acute adverse transfusion
hazards of 214 Hemophilic arthropathy, management of reaction with 252
in transfusion medicine 214 156 complications 261
314  Transfusion Update
hemolysis, pathophysiology of 252 Leucoreduced Myeloma, multiple 3
injury 209 blood, preparation of 222 Myeloproliferative syndromes 3
thrombocytopenic purpura 9 indications for 221
Immunochromatographic 266 packed red blood cells 3 N
Immunoglobulin 3 quality control of 224t National Institute of Immunohaematology
Immunohematology analyzers, fully Leukemia 3 (NIIH) 298
automated 289t Leukocytapheresis 134, 135 Natural killer (NK) cells 211, 212
Immunosuppression, minimize 84 Leukocyte in receptor 212
Incompatibility, major 73 depleted red cells 4 in transplant immunology, role of 212
Indian scenario of ethical issues in reduced blood components 93 in transplantation 211
transfusion medicine 283 Leukodepletion in tumor 211
Infections, acute 19 in blood components 221t origin and distribution of 211
Infectious disease transmission 209, 258 on rejection, impact of 78t response to target cell 211f
Infective shock 256 Leukoencephalopathy 180 Neocytes 141
Inhibitors, management of 156 Leukoreduced blood components, Neonatal
Insulin-like growth factor (IGF) 198, 202 provision of 87 alloimmune thrombocytopenia 116
International Rare Donor Program 297 Ligasure 40 transfusion 67, 69
International Society Lipemia 249 Nerve dysfunction, retinal and optic 150
for Stem Cell Research (ISSCR) 182 Lipoteichoic acid (LTA) 239 Neurological ICU 24
of Blood Transfusion 298 Liver Neurology 199
Code of Ethics 281 biopsy 147 Neutropenia 18
Intracerebral hemorrhage 26, 56 chronic 19
disease
Intracranial pressure 96 classification of 18
chronic end stage 89
Intrauterine transfusion 63, 67, 68 impact of 4
etiology of 89
Intravascular management of isolated 19
iron
coagulation, disseminated 270 Noncardiogenic pulmonary edema (NCPE)
concentration 147
RBC destruction, mechanism of 253 255
content 147, 148
Intravenous immunoglobulin 30, 62, 78 Nonfunction of graft, primary 91
MRI of 148
Iron Nonhealing ulcer 203
Locus control region (LCR) elements 216
accumulation 258 Nonhemolytic febrile 252
Lupus erythematosus 18
chelation 148 Non-Hodgkin lymphoma 209
in nontransfusion dependent Lymphocytic leukemia, development of
Nonimmune causes, acute adverse
thalassemias 152 chronic 209
transfusion reaction with 256
in sickle cell disease and congenital Lymphoma 3
Noninvasive
sideroblastic anemias 152 Lyophilized platelets 33
oxygen measurement 237
chelators, comparison of 149 pH measurement 237
deficiency 208
M
Noninvasive prenatal diagnosis 60
overload 208 Magnetic resonance imaging 148 Nonremunerated blood donors 279
Ischemic stroke 26 Malaria 138, 266 Nonviral delivery systems 215
IVIG, use of 84 Massive transfusion Nucleases 217
IVY method 158, 160 adverse effects of 104 Nucleic acid
definition of 95 amplification
K Megaloblastic anemia 3 assay/techniques 239
Kidney Mesenchymal stem cells (MSCs) 181, 185, techniques 236
disease 189 196 amplification test 230
transplantation 81 Metabolism of iron and erythropoietin 37 targeting 242
Kostmann’s syndrome 3 Methylene blue 243 technology 226
treated platelets 243 test 265
L Meticulous hemostasis 40 vectors 216
Lentiviruses 215 Microcalorimetry 239
Leucocyte Molecular genotyping 292 O
apheresis devices, low 223 Moloney marine leukemia virus (MMLV) OBI, implications of 233
in various red blood cell 215 Occult HBV infection 232
preparations 221 Mono infection 231 Ophthalmology 199
Leucodepletion 225 MTP, complications of 99 Optimization phase (2017) 262
methods of 222 Musculoskeletal pain relief 200f Oral and craniofacial surgeries 199
quality control of 223 Myasthenia gravis 134 Organ dysfunction syndrome, multiple 13
Leucofiltration 222 Mycophenolate mofetil 78 Orthopedic 199
factors affecting 222 Myelodysplastic syndrome 3, 147 liver transplantation 89
of platelet 222 Myelofibrosis, chronic 152 surgery 52
timing of 222 Myeloid growth factor therapy 19 trauma 53
 Index  315

P dose of 112 Pretransfusion testing 67

for neonatal transfusions 69 Propionibacterium spp 237
Packed red
function disorders 127 Progenitor
blood cell 45, 95, 103
gel 199 cell transplant 179
cells 4
increment 5t stem cells 196
Paired kidney donor exchange 81
unit of random donor platelet Prophylactic
Pan genera detection (PGD) 236
transfusion 114t management of patients with liver
test system 239
leukocyte gel 199 disease 165t
Pancreas
mapping 161 platelet transfusion 126
after kidney transplant (PAK) 189
pheresis 136 in dengue 111
alone transplant (PTA) 189
donor 124 Pros and cons of universal leucoreduction
Pancreatic cells 190
poor plasma (PPP) 197-199, 202, 203 224
Pancreatic iron overload, MRI of 148
reasons for discarding 248t Proteolysis of receptors 162
Pancytopenia 18
rich plasma (PRP) 4, 123, 197, 202, 203 Prothrombin
Paraproteinemic polyneuropathies 134
application, preparation of 198 complex concentrate 57, 164
Paroxysmal nocturnal hemoglobinuria 147
applications, timeline for 197f time 111, 158, 159
Partial thromboplastin time, activated 158
in hair transplantation 202 ratio 111
Passenger lymphocyte syndrome 74
in plastic surgery 202 Protozoal infections of red blood cells 137
Pathogen inactivation in fresh frozen
role of 202 Pseudomonas species 256
plasma preparation 243
in regenerative medicine 200 Pseudothrombocytopenia 120
Pathogen reduction 239
role of 197 Public cord blood banking 205
technology (PRT) 242
rationale for 202 Pulmonary embolism 120
Pentoxifylline 194
therapy 197, 198 Pure red cell aplasia 72, 147
Perfluorocarbon emulsions 31
Perfluorochemical compounds 32 use of 203
substitute 33 Q
Peripheral
arterial disease 194 transfusion 68, 112, 114 Qualitative platelet disorders 109
blood stem cell transplant 179 in dengue hemorrhagic fever, criteria Quick’s method 158
Permissive hypotension 40 for 113t
Persistent (chronic) neutropenia, causes indications of 4, 112, 164 R
of 19t triggers 109 Random donor platelets 72, 123
pH 237 utilization guidelines, evidence-based Ransfusion transmitted infection (TTI) 275
Photodynamic method 242 270 Rapid bacterial detection tests, indirect 237
Plasma PLS, management of 74 Rare blood
and platelet compatibility 67 Pluripotent defining 297
derived proteins (PDP) 278 embryonic stem 181 group registry 297
exchange 135 stem (IPS) cells 181, 194 RBC
Plasmodial lactate dehydrogenase 266 Point-of-care tests in cardiac surgery 45 engraftment, delayed 73
Plasmodium Poisoning like nitrobenzene, types of 137 for transfusion, advantages of using 301f
falciparum 138 Polycythemia reduction of graft 73
protozoa 138 primary 136 storage lesions 302t
Plastic surgery 200f secondary 136 transfusion 3
Platelet 14, 104, 164 Polymerase chain reaction (PCR) 226 in cancer, impact of 209
aggregation 238 Polymyositis 134 RCC and whole blood, comparison between
apheresis 136 Pooled buffy coat method, advantages of 4t
causes of destruction of 108f 123 Reactions of transfusions 104
concentrate (PCs) 4, 236, 242, 305 Porphyria cutanea tarda 135 Reagent red cells, quality control in 294
preparation of 123 Portal pressure, reduction of 164 Recipient cytokines 224
with amotosalen photochemical Positive direct antiglobulin test 93 Red blood cells (RBCs) 9, 20, 24, 37, 138,
treatment 242 Poststorage filtration 223 198, 289, 301
count 158, 160 Post-transfusion purpura 120, 209, 257, 260 exchange 137
monitoring 119 Potassium effects 257 indications of 137
for HIT8 120t Potential benefits of leucoreduction 221 technique 137
defect 157 Practical considerations of TP 131 short-term 15
derived Predonation of autologous blood 52 transfusion 24, 164
epidermal growth factor 198 Pre-existing coagulopathy 90 role of 24
growth factor (PDGF) 198, 202 Premature termination codon suppression utilization guidelines, evidence-based
microparticles 33 155 270
products 33 Prenatal testing 63t Red cell
disorders 109 Prestorage filtration 222 concentrates 3
acquired 108 Prestorage leucodepletion vs poststorage preparations 68
inherited 108 leucodepletion 223t substitutes 31
316  Transfusion Update
transfusion 208, 270 Splenectomy 82, 144 leucapheresis 135
indications of 110 Squid 147 plasma exchange (TPE) 133, 134, 243
units in patients with Allo antibodies, Staphylococcus 236 platelet products 199
selection of 92 State Blood Transfusion Council (SBTC) plateletpheresis 131
Regenerative medicine 197 276 orders 131
integrity of 182 Stem cell 195, 204 Thrombin 164
Reinvigorate dormant hair follicles 203 action of 184 time (TT) 158, 159
Renal failure, chronic 297 delivery, methods of 185 Thrombocytapheresis 136
Respiratory disease 308 ethical issues with use of 191 Thrombocytopenia 16, 17, 91, 108
Retroviruses 215 graft infusion 73 arises from causes 16
Revision hip replacement 49 in regenerative medicine 181 chronic stable 126
RHD prolotherapy 197 treatment of 18
gene, partial variants of 293 science into regenerative medicine, with absent radii 3
in fetus, prenatal diagnosis of 293 translation of 181 with anemia and leucopenia 18
zygosity testing 293 technology 194 Thrombocytosis and rationale for
Ribonucleic acid (RNA) 226 therapy 195 therapeutic plateletpheresis (TP)
Right to refusal 283 for cardiac repair 185 131
Rituximab 82, 83 for diabetes 190 Thromboelastograph hemostasis analyzer
Robotic surgery 41f mellitus 189 (TEG) 160
Rotem 161 for pad 194 Thromboelastography (TEG) 42, 99, 160
in acute myocardial infarction 186 Thromboelastometry 99
S in cardiovascular diseases 184 Thrombolytic therapy, reversal of 174
SD plasma in chronic ischemic heart failure 186 Thromboplastin time 110
production of 171 in dilated cardiomyopathy 186 Thrombotic thrombocytopenic purpura 271
properties of 171 in PAD, current status of 196 Tissue
use of 172t transplant, results of 186 healing 203
Serine protease inhibitor 164 transplantation 144, 179, 191 repair 203
Serious hazards of transfusion (SHOT) 305 type of 190 Total hip replacement, primary 48
Serologic testing, automation in 288 Steroids in treatment of AIHA, role of 8 Total knee arthroplasty, primary 49
Serological versus molecular assays 264 Streptococcus pyogenes 9 Toxins 3
Seropositivity reason for discarding Strepyomyces pilosus 149 Tranexamic acid 49, 99, 164
FFP 248t Stroke 154 Transcutaneous oxygen tension 196
platelets 247t Subarachnoid hemorrhage 26, 56 Transepicardial injection 186
Seroprevalence in India 232 Sugar free antibodies 9 Transforming growth factor 198
Serratia liquefaciens 237 in autoimmune diseases 11 beta (TGF-b) 202
Serum ferritin 147 in autoimmune hemolytic anemia 9 Transfused white blood cells, effects of 72
Sex determination 61 in immune thrombocytopenic purpura 9 Transfusion 7, 15, 74, 127
Shwachman-Diamond syndrome (SDS) 3 Suicide gene 215 adverse effects of 25
Sickle cell Superconducting quantum interference and iron chelation 147t
anemia 214, 217, 297 device 147 associated circulatory overload (TACO)
disease (SCD) 136, 137, 147, 256 Surgical blood order schedule 48 51
Sideroblastic anemia 147 Surgical control of hemorrhage 95 associated graft versus host disease 258,
Simultaneous kidney pancreas Syndrome, small-for-size 90 301
transplant 189 Synthesis of coagulation factors 162t associated infections 145
Single donor platelet (SDP) 72, 123 Synthetic oxygen carriers 5 complications of 208
plateletpheresis 124 Synthetic platelet alternatives 33 in extracorporeal membrane oxygenation
versus random donor platelets 123 Syphilis serology 265 (ECMO) 70
Single-nucleotide polymorphism (SNP) 299 in necrotizing enterocolitis (NEC) 70
Skeletal T in neuro-ICU 25
changes 150 Tachycardia 3 in obstetrical hemorrhage, massive 102
myoblasts 185 Teratogenicity 151 in renal transplantation, evolution of 77
injection of 186 Thalassemia 143, 297 in sickle cell anemia 153
Skin complications of 144 interval 141
rash 151 intermedia 143, 147 massive 174
rejuvenation 203 major 143, 147 medicine 292
Solid-phase immunoassays 288 minor 143 elements of evidence-based 269f
Solid-phase red cell adherence (SPRCA) transfusion programs, problem of 142 ethical issues in 281
289 Therapeutic evidence-based 269, 271
Solvent detergent treated plasma in clinical cytapheresis 135 of allogenic blood 236
use 171 dose 109 of preterm infants 68
Somatic gene therapy 214 erythrocytapheresis 135, 136 of products 106
 Index  317

of red cells, exchange 67 risk scoring system 45t V

practice in arthroplasty 48 sepsis 237 Vascular endothelial growth factor (VEGF)
practice in support 92 194, 198, 202
bone marrow transplantation and in cardiac surgery 44 Vasculitides and ANCA disorders 134
hla matching 71 in neurosurgery 55 Vector design 215
coronary interventions 20 therapy 140, 141 Veno-occlusive liver disease 180
disseminated intravascular in autoimmune hemolytic anemia 7 Venous thromboembolism 172
coagulation 13 transmitted bacterial infections (TTBI) Ventricular wall 185
heart transplant 87, 89 236 Vessel wall defect 157
practice in transmitted infections (TTIs) 71, 226, 230 Viral
oncology 15 levels of 282 delivery systems 215
renal transplants recipients 76 triggers 126 infections 18, 168
thalassemia 140 variability in cardiac surgery 44 ribonucleic acid (RNA) 230
practices 71 with circulatory overload 208 Vitamin K 30, 164, 165
predictors 89 Transient (acute) neutropenia, causes of 18 von Willebrand’s
protocol disease 174, 271
Transjugular intrahepatic portosystemic
adult, massive 97t factor 243
shunt (TIPSS) 90
during liver transplantation 93
Transvascular route 185
in minor ABO mismatched liver
Traumatic brain injury 25, 56 W
transplants 93
severe 24 Warm antibody autoimmune hemolytic
massive 95, 98t, 102
Treponema pallidum 301 anemia 8
reaction 5
TT HBV with OBI, risk of 233 Warming blood 305
adverse 252
Tumor Washed packed RBCs 141
classification, adverse 252
progression 208 Whole blood
definition of adverse 252
surgery 56 derived platelets 123
reporting form 262
transfusion, indications of 35
with immune causes, adverse 257
without immune causes, adverse 258 U Wound healing 203
chronic 199, 200f
regimen 141 Ultraviolet light treated platelets 243
related acute lung injury (TRALI) 5, 25, Umbilical blood sampling 63 Y
51, 209, 222, 255, 255f, 260, 302 Unfractionated heparin (UFH) 122 Yersinia enterocolitica 236, 256
related graft vs host disease 209 Universal leucodepletion vs selective
related immunomodulation (TRIM) 21,
leucodepletion 224 Z
51
Uremia 109 Zinc
related risks 5
Uremic coagulopathy 174 deficiency 151
requirements
Urticarial reactions 5, 255 finger nuclease (ZFN) 217
during transplant 87
in critical care 51